General Strategy for Specific Fluorescence Imaging of Homocysteine in Living Cells and In Vivo.

The aberrantly changed level of homocysteine (Hcy) triggers a variety of pathological symptoms and subsequently Hcy-related diseases. Direct and selective visualization of Hcy in biological systems is pivotal to understanding the pathological functions of Hcy at the molecular level. Herein, a general strategy was developed for the specific fluorescence imaging of Hcy through the combination of dual-binding sites and the introduction of a nitro group at the 6-position of the 7-diethylaminocoumarin fluorophore. Also, a series of novel fluorescent probes were exploited for monitoring Hcy with excellent selectivity, high sensitivity, and far-red/near-infrared fluorescence emission. Furthermore, fluorescence imaging of endogenous Hcy dynamics in living cells and in vivo was achieved, providing direct and solid evidence for the increasement of endogenous Hcy in type 2 diabetes mellitus and Alzheimer's disease. This research will greatly advance the development and understanding of the molecular nexus between the Hcy metabolism cascade and the root causes of diseases related to Hcy.

A large Stokes shift NIR fluorescent probe for visual monitoring of mitochondrial peroxynitrite during inflammation and ferroptosis and in an Alzheimer's disease model.

The excessive formation of peroxynitrite (ONOO-) in mitochondria has been implicated in various pathophysiological processes and diseases. However, owing to short emission wavelengths and small Stokes shifts, previously reported fluorescent probes pose significant challenges for mitochondrial ONOO- imaging in biological systems. In this study, a near-infrared (NIR) fluorescent probe, denoted as DCO-POT, is designed for the visual monitoring of mitochondrial ONOO-, displaying a remarkable Stokes shift of 170 nm. The NIR fluorophore of DCO-CHO is released by DCO-POT upon the addition of ONOO-, resulting in off-on NIR fluorescence at 670 nm. This phenomenon facilitates the high-resolution confocal laser scanning imaging of ONOO- generated in biological systems. The practical applications of DCO-POT as an efficient fluorescence imaging tool are verified in this study. DCO-POT enables the fluorometric visualization of ONOO- in organelles, cells, and organisms. In particular, ONOO- generation is analyzed during cellular and organism-level (zebrafish) inflammation during ferroptosis and in an Alzheimer's disease mouse model. The excellent visual monitoring performance of DCO-POT in vivo makes it a promising tool for exploring the pathophysiological effects of ONOO-.

A novel AIE fluorescent probe for the monitoring of aluminum ions in living cells and zebrafish.

A novel fluorescent probe BTD with aggregation induced emission (AIE) characteristics for the monitoring of Al3+ was developed. This fluorescent probe could be used to detect Al3+ in aqueous solution under mild conditions, along with high sensitivity and high selectivity. The detection limit of the probe BTD for Al3+ is as low as 3.25 nM, which is below the WHO recommendation concentration (7.41 µM) for drinking water. Furthermore, this probe was successfully applied to the sensing of Al3+ in living cells and zebrafish.

Direct Quantification and Visualization of Homocysteine, Cysteine, and Glutathione in Alzheimer's and Parkinson's Disease Model Tissues.

Alzheimer's disease (AD) and Parkinson's disease (PD) are chronic neurodegenerative diseases with high morbidity and mortality. Homocysteine (Hcy), cysteine (Cys), and glutathione (GSH) are closely related to AD and PD. However, the dynamics of Hcy, Cys, and GSH in the brain tissues and the potential pathogenesis between Cys/Hcy/GSH with AD and PD remain unclear. Herein, a novel fluorescent probe 1 with multiple binding sites was rationally designed and exploited for the direct quantification of serum total Hcy and Cys along with superior optical properties. Importantly, differentiation and simultaneity fluorescence imaging of Cys, Hcy, and GSH dynamics were achieved in living cells, tissues, and mouse models of AD and PD with this probe, providing direct evidences for the relationship between Hcy/Cys/GSH and AD/PD for the first time. In addition, pathogenesis studies demonstrated that elevated Hcy and Cys levels are closely related to imbalanced redox homeostasis, increased amyloid aggregates, and nerve cell cytotoxicity. These findings will greatly promote the understanding of the functions of Hcy/Cys/GSH in Alzheimer's and Parkinson's diseases, demonstrating clinical promise for the early diagnosis and prevention of AD and PD.

A novel ratiometric fluorescent probe for the detection of mitochondrial pH dynamics during cell damage.

A sensitive fluorescent probe (E)-4-(3-(benzo[d]thiazol-2-yl)-4-hydroxy-5-methylstyryl)-1-methylpyridin-1-ium iodide (HBTMP) for the monitoring of pH in mitochondria was rationally exploited. This novel probe exhibited remarkable pH-dependent behavior in the linear range of 5.5-8.0, along with a pKa value of 6.829 ± 0.02627. A large Stokes shift of 205 nm was obtained. This fluorescent probe demonstrated good biocompatibility and high sensitivity for detecting the dynamic changes in mitochondrial pH in living cells and zebrafish. The results of the CCCP (m-chlorophenyl hydrazone) treatment experiment indicated that the probe can effectively monitor changes in mitochondrial pH caused by cell damage.

Molecular systematics of Pampus (Perciformes: Stromateidae) based on thousands of nuclear loci using target-gene enrichment.

Pomfret fishes of the genus Pampus are commercially important in the Indo-Pacific region, yet the phylogenetic relationships and taxonomy of Pampus remain contentious. Here, we sampled 151 specimens, representing all known species of the genus, as well as two outgroup species (two families). We collected sequences from 17,292 single-copy nuclear coding loci using target-gene enrichment and Illumina sequencing for a subset samples of P. echinogaster, P. argenteus, P. cinereus, P. liuorum, P. chinensis, P. minor, and P. punctatissimus, which were carefully examined according to their species descriptions. Concatenated gene tree and species tree analyses resulted in identical and highly supported phylogenies, in which P. argenteus was sister to P. minor in one clade and P. cinereus sister to P. chinensis and P. punctatissimus in the other clade. Phylogenetic reconstruction using sequences of cytochrome c oxidase subunit I (COI) collected by us and those retrieved from NCBI suggests extensive misidentification of Pampus species in the NCBI database. We also measured morphological characters of each species as well as observed their osteological structure using micro-CT. Both molecular and morphological results suggest that P. echinogaster is a synonym of P. argenteus, and P. liuorum is a synonym of P. cinereus. Pampus cinereus from China, Bangladesh and an uncertain origin were grouped into three clades according to their sampling localities, but we could not find decisive morphological characters to describe the "cryptic species" of P. cinereus. Finally, based on the results of the molecular analyses and morphological reexamination, we created an identification key for the genus of Pampus.

A novel colorimetric and ratiometric fluorescent probe for sensing SO2 derivatives and their bio-imaging in living cells.

We report a novel fluorescent probe HBN-TCF for the detection of SO2 derivatives. This probe exhibited near-infrared fluorescence emission with an excitation wavelength of 620 nm. After reacting with SO32-, the emission channel at 664 nm decreased, while the new strong emission channel at 482 nm increased (λex = 400 nm), with a large emission distance (Δλ = 182 nm) observed. This probe exhibited the rapid and selective detection of SO2 derivatives compared with other sulfur-containing species and featured a low detection limit (82 nM). This colorimetric and ratiometric fluorescent probe showed high selectivity and sensitivity for detecting SO2 derivatives. The probe was also successfully exploited for the fluorescence imaging of intracellular and exogenous SO2 derivatives in BEL-7402 cells.

A dual-responsive colorimetric probe for the detection of Cu2+ and Ni2+ species in real water samples and human serum.

The monitoring of heavy transition metals has increasingly attracted great attention because they pollute the environment and have unique physiological functions. Chemosensors are useful tools for monitoring heavy transition metals due to their simple visualization, excellent sensitivity and high selectivity. Herein, we have developed a novel chemosensor for the detection of water-soluble Cu2+ and Ni2+ species with different mechanisms, and low detection limits of 2.1 nM for Cu2+ and 1.2 nM for Ni2+ were obtained. The colorimetric probe CPH has been applied to qualitative and quantitative detection of Cu2+ and Ni2+ species in real samples.

Simultaneous Visualization of Endogenous Homocysteine, Cysteine, Glutathione, and their Transformation through Different Fluorescence Channels.

Studying numerous biologically important species simultaneously is crucial to understanding cellular functions and the root causes of related diseases. Direct visualization of endogenous biothiols in biological systems is of great value to understanding their biological roles. Herein, a novel multi-signal fluorescent probe was rationally designed and exploited for the simultaneous sensing of homocysteine (Hcy), cysteine (Cys), and glutathione (GSH) using different emission channels. This probe was successfully applied to the simultaneous discrimination between and visualization of endogenous Hcy, Cys, GSH, and their transformation in living cells.

A Multi-signal Fluorescent Probe with Multiple Binding Sites for Simultaneous Sensing of Cysteine, Homocysteine, and Glutathione.

A novel fluorescent probe was developed by integrating chlorinated coumarin and benzothiazolylacetonitrile and exploited for simultaneous detection of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Featuring four binding sites and different reaction mechanisms for different biothiols, this probe exhibited rapid fluorescence turn-on for distinguishing Cys, Hcy, and GSH with 108-, 128-, 30-fold fluorescence increases at 457, 559, 529 nm, respectively, across different excitation wavelengths. Furthermore, the probe was successfully applied to the fluorescence imaging of endogenous Cys and GSH and exogenous Cys, Hcy, and GSH in living cells.

Critically short telomeres derepress retrotransposons to promote genome instability in embryonic stem cells.

Telomeres, at the ends of chromosomes, protect chromosomes from fusion and preserve genomic stability. However, the molecular mechanisms underlying telomere attrition-induced genome instability remain to be understood. We systematically analyzed the expression of retrotransposons and performed genomic sequencing of different cell and tissue types with telomeres of varying lengths due to telomerase deficiency. We found that critically short telomeres altered retrotransposon activity to promote genomic instability in mouse embryonic stem cells, as evidenced by elevated numbers of single nucleotide variants, indels and copy number variations (CNVs). Transpositions of retrotransposons such as LINE1 resulting from the short telomeres can also be found in these genomes with elevated number of mutations and CNVs. Retrotransposon activation is linked to increased chromatin accessibility, and reduced heterochromatin abundance correlates with short telomeres. Re-elongation of telomeres upon recovery of telomerase partly represses retrotransposons and heterochromatin accumulation. Together, our findings suggest a potential mechanism by which telomeres maintain genomic stability by suppressing chromatin accessibility and retrotransposon activity.

Culture conditions of mouse ESCs impact the tumor appearance in vivo.

Various culture conditions by small molecules have been explored to extend pluripotency of stem cells, but their impacts on cell fate in vivo remain elusive. We systematically compared the effects of various culture conditions on the pluripotency and cell fate in vivo of mouse embryonic stem cells (ESCs) by tetraploid embryo complementation assay. Conventional ESC cultures in serum/LIF-based condition produced complete ESC mice and also the survival to adulthood at the highest rates of all other chemical-based cultures. Moreover, long-term examination of the survived ESC mice demonstrated that conventional ESC cultures did not lead to visible abnormality for up to 1.5-2 years, whereas the prolonged chemical-based cultures developed retroperitoneal atypical teratomas or leiomyomas. The chemical-based cultures exhibited transcriptomes and epigenomes that typically differed from those of conventional ESC cultures. Our results warrant further refinement of culture conditions in promoting the pluripotency and safety of ESCs in future applications.

Simultaneous sensing of cysteine/homocysteine and glutathione with a fluorescent probe based on a single atom replacement strategy.

In recent years, there have been many reports of fluorescent probes for multi-channel detection of Cys, Hcy and GSH. Particularly, reports of fluorescent probes using NBD (7-nitro-1,2,3-benzoxadiazole) or SNBD (7-nitro-1,2,3-benzothiadiazole) moieties as fluorophores are particularly common. Unfortunately, their 4-sulfhydryl derivatives exhibited negligible fluorescence, which makes them incapable of detecting GSH directly. Herein, by performing single selenium-for-oxygen atom replacement within 4-chloro-substituted NBD (NBD-Cl), we developed a small molecule fluorescent probe based on a single atom replacement strategy, which enables the probe to be used for simultaneously distinguishing Cys/Hcy and GSH, along with fluorescence imaging of Cys/Hcy and GSH in live cells from red and green emission channels, respectively.

Turn-on fluorescent probe for sensing exogenous and endogenous hypochlorous acid in living cells, zebrafishes and mice.

Hypochlorous acid (HOCl), belonging to biologically significant reactive oxygen species (ROS), plays crucial roles in many biological and pathological processes. It is of great value to explore fluorescent probes for the image of hypochlorous acid in various biological environments. We herein reported a novel fluorescent probe HN-ClO for monitoring HOCl with moderate water-solubility, good photostability, high fluorescence quantum yield and large Stokes shift. This probe exhibited excellent selectivity and high sensitivity to sense HOCl. Furthermore, probe HN-ClO was successfully applied to monitor endogenous and exogenous HOCl in living cells, zebrafishes and mice, and possessed the potential to further explore the physiological and pathological roles of hypochlorous acid in biological systems.

A novel red emission fluorescent probe for monitoring carbon monoxide in living cells and zebrafish.

Carbon monoxide (CO), a gaseous signal molecule, plays a crucial role in biological systems. With the aim of unraveling its biological functions, a novel fluorescent probe for sensing CO was rationally designed and synthesized based on a coumarin derivative fluorophore merging tetrahydroquinoxaline unit and five-membered pyrrolidine. This fluorescent probe demonstrated a large Stokes shift (Δλ = 132 nm), high quantum yield, red emission, high sensitivity and selectivity for CO with remarkable fluorescence turn-on. And the detection limit for CORM-3 is as low as 31.2 nM with the linear range of 0-30 µM. More importantly, this novel probe has been successfully applied to the fluorescence imaging of CO in HepG2 cells and zebrafish, providing a useful approach for the further understanding of the physiological and pathological roles of CO in living systems.

Cross-species gene enrichment revealed a single population of Hilsa shad (Tenualosa ilisha) with low genetic variation in Bangladesh waters.

Tenualosa ilisha is a popular anadromous and significant trans-boundary fish. For sustainable management and conservation of this fish, drawing an appropriate picture reflecting population status of this species is very essential based on their all-strategic habitats in Bangladesh. In this study, 139 samples from 18 sites were collected and cross-species gene enrichment method was applied. Like most of the Clupeiforms, nucleotide diversity of this shad was very low (0.001245-0.006612). Population differences between most of the locations were low and not significant (P > 0.05). However, P values of a few locations were significant (P < 0.05) but their pairwise FST values were very poor (0.0042-0.0993), which is inadequate to recognize any local populations. Our study revealed that the presence of a single population in the Bangladesh waters with some admixtured individuals, which may contain partial genes from other populations. Most of the individuals were admixed without showing any precise grouping in the ML IQtree and Network, which might due to their highly migratory nature. Fishes from haors and small coastal rivers were not unique and no genetic differences between migratory cohorts. The hilsa shad fishery should be managed considering it as a single panmictic population in Bangladesh with low genetic diversity.

A novel fluorescent probe for selective imaging of cellular cysteine with large Stokes shift and high quantum yield.

A new strategy endowing probe with large Stokes shift, high quantum yield and red emission was developed by incorporating tetrahydroquinoxaline unit and five-membered pyrrolidine ring on fluorophore. As demonstrated, a novel probe was rationally designed and synthesized for sensitive monitor of cellular cysteine (Cys) and endogenous aminoacylase with large Stokes shift and high quantum yield.

A novel fluorescent probe for detection of Glutathione dynamics during ROS-induced redox imbalance.

Monitoring cellular GSH dynamics is of great value to understand its complex biological functions and related diseases. It still remains challenging to understand the mechanism of GSH dynamics in complex intracellular environment. Herein, a novel fluorescent probe featured with chlorinated coumarin-TCF was exploited for sensing of GSH with high selectivity and high sensitivity. Large fluorescence enhancement at 471 nm was observed upon addition of GSH with large emission distance (Δλem = 219 nm). This novel probe was successfully applied to monitor endogenous and exogenous GSH dynamics without interference of other thiols via fluorescence imaging. More importantly, using this probe, low dose reactive oxygen species induced GSH increasement through nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signal pathway in BEL-7402 cells was observed. This novel fluorescent probe has the potential for quantitative monitoring of cellular GSH, which will promote further understanding of its physiological and pathological roles in biological systems.

A novel fluorescent probe with dual-sites for simultaneously monitoring metabolisms of cysteine in living cells and zebrafishes.

Understanding cellular metabolism holds immense potential for developing new drugs that regulate metabolic pathways. Two gas signal molecules, SO2 and H2S, are the main metabolites from cysteine (Cys) via oxidation and desulfurization pathways, respectively. However, a few fluorescent probes for real-time monitor of the metabolic pathways of cysteine have been reported. To understand metabolic alterations of cysteine, we have rationally designed and prepared a dual-signal fluorescent probe HN, which could differentiate SO2 and H2S through two different fluorescence channels simultaneously, along with similar reaction kinetics and both "off-on" fluorescence responses. Probe HN exhibits the potential to monitor the metabolism pathways of cysteine, and the distinguishment of cancer cells from normal cells could be realized. This methodology will promote further understanding of the physiological and pathological roles of cysteine.

A dual-emission and mitochondria-targeted fluorescent probe for rapid detection of SO2 derivatives and its imaging in living cells.

A novel mitochondria-targeted fluorescent probe for sensing SO2 derivatives was constructed by integrating phenanthroimidazole and cyanine moieties. Excited by different channels, this probe exhibited unique dual-channel fluorescence-enhanced emissions and colorimetric response to HSO3- quickly (less than 20 s). The sensing process was confirmed to undergo nucleophilic addition of HSO3- to C˭C double bond in the probe Mito-PTB via NMR and HRMS analyses. Furthermore, fluorescence co-localization studies demonstrated that the probe Mito-PTB was a mitochondria-targetable fluorescent probe for SO2 derivatives with good cell membrane permeability. Thus, probe Mito-PTB has the potential applications to explore the role played by SO2 derivatives in biology.