Advances in small-molecule fluorescent probes for the study of apoptosis.

Apoptosis, as type I cell death, is an active death process strictly controlled by multiple genes, and plays a significant role in regulating various activities. Mounting research indicates that the unique modality of cell apoptosis is directly or indirectly related to different diseases including cancer, autoimmune diseases, viral diseases, neurodegenerative diseases, etc. However, the underlying mechanisms of cell apoptosis are complicated and not fully clarified yet, possibly due to the lack of effective chemical tools for the nondestructive and real-time visualization of apoptosis in complex biological systems. In the past 15 years, various small-molecule fluorescent probes (SMFPs) for imaging apoptosis in vitro and in vivo have attracted broad interest in related disease diagnostics and therapeutics. In this review, we aim to highlight the recent developments of SMFPs based on enzyme activity, plasma membranes, reactive oxygen species, reactive sulfur species, microenvironments and others during cell apoptosis. In particular, we generalize the mechanisms commonly used to design SMFPs for studying apoptosis. In addition, we discuss the limitations of reported probes, and emphasize the potential challenges and prospects in the future. We believe that this review will provide a comprehensive summary and challenging direction for the development of SMFPs in apoptosis related fields.

Cascading Detection of Hydrogen Sulfide and N-Acetyltransferase 2 in Hepatocellular Carcinoma Cells Using a Two-Photon Fluorescent Probe.

Hydrogen sulfide (H2S), a critical gas signaling molecule, and N-acetyltransferase 2 (NAT2), a key enzyme in drug metabolism, are both known active biomarkers for liver function. However, the interactions and effects of H2S and NAT2 in living cells or lesion sites remain unknown due to the lack of imaging tools to achieve simultaneous detection of these two substances, making it challenging to implement real-time imaging and precise tracking. Herein, we report an activity-based two-photon fluorescent probe, TPSP-1, for the cascade detection of H2S and NAT2 in living liver cells. Continuous conversion from TPSP-1 to TPSP-3 was achieved in liver cells and tissues. Significantly, leveraging the outstanding optical properties of this two-photon fluorescent probe, TPSP-1, has been effectively used to identify pathological tissue samples directly from clinical liver cancer patients. This work provides us with this novel sensing and two-photon imaging probe, which can be used as a powerful tool to study the physiological functions of H2S and NAT2 and will help facilitate rapid and accurate diagnosis and therapeutic evaluation of hepatocellular carcinoma.

Promoting In Vivo NIR-II Fluorescent Imaging for Lipid in Lipid Metabolism Diseases Diagnosis.

Lipid metabolism diseases have become a tremendous risk worldwide, along with the development of productivity and particular attention to public health. It has been an urgent necessity to exploit reliable imaging strategies for lipids and thus to monitor fatty liver diseases. Herein, by converting the NIR-I signal to the NIR-II signal with IR1061 for the monitoring of lipid, the in vivo imaging of fatty liver disease was promoted on the contrast and visual effect. The main advantages of the imaging promotion in this work included a long emission wavelength, rapid response, and high signal-background-ratio (SBR) value. After promoting the NIR-I signal to NIR-II signal, IR1061 achieved higher SBR value and exhibited a dose-dependent fluorescence intensity at 1100 nm along with the increase of the EtOH proportion as well as steady and selective optical responses toward liposomes. IR1061 was further applied in the in vivo imaging of lipid in fatty liver diseases. In spite of the differences in body weight gain and TC level between healthy mice and fatty liver diseases two models, IR1061 achieved high-resolution imaging in the liver region to monitor the fatty liver disease status. This work might be informatic for the clinical diagnosis and therapeutical treatments of fatty liver diseases.

In situ imaging of signaling molecule carbon monoxide in plants with a fluorescent probe.

Carbon monoxide (CO) is a recently discovered gasotransmitter. In animals, it has been found that endogenously produced CO participates in the regulation of various metabolic processes. Recent research has indicated that CO, acting as a signaling molecule, plays a crucial regulatory role in plant development and their response to abiotic stress. In this work, we developed a fluorescent probe, named COP (carbonic oxide Probe), for the in situ imaging of CO in Arabidopsis thaliana plant tissues. The probe was designed by combining malononitrile-naphthalene as the fluorophore and a typical palladium-mediated reaction mechanism. When reacted with the released CO, COP showed an obvious fluorescence enhancement at 575 nm, which could be observed in naked-eye conditions. With a linear range of 0-10 µM, the limit of detection of COP was determined as 0.38 µM. The detection system based on COP indicated several advantages including relatively rapid response within 20 min, steadiness in a wide pH range of 5.0-10.0, high selectivity, and applicative anti-interference. Moreover, with a penetration depth of 30 µm, COP enabled 3D imaging of CO dynamics in plant samples, whether it was caused by agent release, heavy metal stress, or inner oxidation. This work provides a fluorescent probe for monitoring CO levels in plant samples, and it expands the application field of CO-detection technology, assisting researchers in understanding the dynamic changes in plant physiological processes, making it an important tool for studying plant physiology and biological processes.

Grid composite meta-surface absorber with thermal isolation structure for terahertz detection.

This paper specifically focuses on the absorber, the critical component responsible for the detector's response performance. The meta-surface absorber combines two resonant structures and achieves over 80% absorptance around 210 GHz, resulting in a broad operating frequency range. FR-4 is selected as the dielectric layer to be compatible with standard printed circuit board (PCB) technology, which reduces the overall fabrication time and cost. The absorbing unit and array layout are symmetrically designed, providing stable absorptance performance even under incident waves of different polarization angles. The polarization-insensitive absorptance characteristic further enhances the compatibility between the absorber and the detector in the application scenario. Furthermore, the thermal insulation performance of the absorber is ensured by introducing thermal insulation gaps. After completing fabrication through PCB technology, testing revealed that the absorber maintained excellent absorptance performance within its primary operating frequency range. This performance consistency closely matched the simulation results.

A human serum albumin-binding-based fluorescent probe for monitoring hydrogen sulfide and bioimaging.

In this work, a fluorescent probe, TPABF-HS, was developed for detecting hydrogen sulfide (H2S) using a human serum albumin (HSA)-binding-based approach for amplifying the fluorescence signal and extending the linear correlation range. Compared to the most recent probes for H2S, the most interesting feature of the detection system developed herein was the especially wide linear range (0-1000 µM (0-100 eq.)), which covered the physiological and pathological levels of H2S. TPABF-HS could be used in applications high sensitivity and selectivity with an LOD value of 0.42 µM. Further, site-competition experiments and molecular docking simulation experiments indicated that signal amplification was realized by the binding of the TPABF fluorophore to the naproxen-binding site of HSA. Moreover, the extension of the measurement span could allow for applications in living cells and Caenorhabditis elegans for imaging both exogenous and endogenous H2S. This work brings new information to the strategy of signal processing by exploiting fluorescent probes.

Real-time monitoring of abnormal mitochondrial viscosity in glioblastoma with a novel mitochondria-targeting fluorescent probe.

Glioblastoma is the most fatal and insidious malignancy, due to the existence of the blood-brain barrier (BBB) and the high invasiveness of tumor cells. Abnormal mitochondrial viscosity has been identified as a key feature of malignancies. Therefore, this study reports on a novel fluorescent probe for mitochondrial viscosity, called ZVGQ, which is based on the twisted intramolecular charge transfer (TICT) effect. The probe uses 3-dicyanomethyl-1,5,5-trimethylcyclohexene as an electron donor moiety and molecular rotor, and triphenylphosphine (TPP) cation as an electron acceptor and mitochondrial targeting group. ZVGQ is highly selective, pH and time stable, and exhibits rapid viscosity responsiveness. In vitro experiments showed that ZVGQ could rapidly recognize to detect the changes in mitochondrial viscosity induced by nystatin and rotenone in U87MG cells and enable long-term imaging for up to 12 h in live U87MG cells. Additionally, in vitro 3D tumor spheres and in vivo orthotopic tumor-bearing models demonstrated that the probe ZVGQ exhibited exceptional tissue penetration depth and the ability to penetrate the BBB. The probe ZVGQ not only successfully visualizes abnormal mitochondrial viscosity changes, but also provides a practical and feasible tool for real-time imaging and clinical diagnosis of glioblastoma.

Synthesis and biological evaluation of triazolones/oxadiazolones as novel urease inhibitors.

Urease is the main virulence factor of infectious gastritis and gastric ulcers. Urease inhibitors are regarded as the first choice for the treatment of such diseases. Based on the triazolone/oxadiazolone skeleton, a urea-like fragment being able to specifically bind the urease activity pocket and prevent urea from hydrolysis, we designed and synthesized 45 triazolones/oxadiazolones as urease inhibitors. Eight compounds were proved to show excellent inhibitory activity against Helicobacter pylori urease, being more potency than the clinically used urease inhibitor acetohydroxamic acid. The most active inhibitor with IC50 value of 1.2 µM was over 20-fold higher potent than the positive control. Enzymatic kinetic assays showed that these novel inhibitors reversibly inhibited urease with a mixed competitive mechanism. Molecular dockings provided evidence for the observations in enzyme assays. Furthermore, these novel inhibitors were proved as drug-like compounds with very low cytotoxicity to mammalian cells and favorable water solubility. These results suggested that triazolone and oxadiazolone were promising scaffolds for the design and discovery of novel urease inhibitors, and were expected as good candidates for further drug development.

Potassium sodium hydrogen citrate intervention on gut microbiota and clinical features in uric acid stone patients.

The high recurrence rate of renal uric acid stone (UAS) poses a significant challenge for urologists, and potassium sodium hydrogen citrate (PSHC) has been proven to be an effective oral dissolution drug. However, no studies have investigated the impact of PSHC on gut microbiota and its metabolites during stone dissolution therapy. We prospectively recruited 37 UAS patients and 40 healthy subjects, of which 12 patients completed a 3-month pharmacological intervention. Fasting vein blood was extracted and mid-stream urine was retained for biochemical testing. Fecal samples were collected for 16S ribosomal RNA (rRNA) gene sequencing and short chain fatty acids (SCFAs) content determination. UAS patients exhibited comorbidities such as obesity, hypertension, gout, and dyslipidemia. The richness and diversity of the gut microbiota were significantly decreased in UAS patients, Bacteroides and Fusobacterium were dominant genera while Subdoligranulum and Bifidobacterium were poorly enriched. After PSHC intervention, there was a significant reduction in stone size accompanied by decreased serum uric acid and increased urinary pH levels. The abundance of pathogenic bacterium Fusobacterium was significantly downregulated following the intervention, whereas there was an upregulation observed in SCFA-producing bacteria Lachnoclostridium and Parasutterella, leading to a significant elevation in butyric acid content. Functions related to fatty acid synthesis and amino acid metabolism within the microbiota showed upregulation following PSHC intervention. The correlation analysis revealed a positive association between stone pathogenic bacteria abundance and clinical factors for stone formation, while a negative correlation with SCFAs contents. Our preliminary study revealed that alterations in gut microbiota and metabolites were the crucial physiological adaptation to PSHC intervention. Targeted regulation of microbiota and SCFA holds promise for enhancing drug therapy efficacy and preventing stone recurrence. KEY POINTS: • Bacteroides and Fusobacterium were identified as dominant genera for UAS patients • After PSHC intervention, Fusobacterium decreased and butyric acid content increased • The microbiota increased capacity for fatty acid synthesis after PSHC intervention.

Development of a hypoxia-activated red-emission fluorescent probe for in vivo tumor microenvironment imaging and anti-tumor therapy.

Hypoxia, a significant feature of the tumor microenvironment, is closely associated with tumor growth, metastasis, and drug resistance. In the field of tumor microenvironment analysis, accurately imaging and quantifying hypoxia - a critical factor associated with tumor progression, metastasis, and resistance to therapy - remains a significant challenge. Herein, a hypoxia-activated red-emission fluorescent probe, ODP, for in vivo imaging of hypoxia in the tumor microenvironment is presented. Among various imaging methods, optical imaging is particularly convenient due to its rapid response and high sensitivity. The ODP probe specifically targets nitroreductase (AzoR), an enzyme highly expressed in hypoxic cells, playing a vital role by catalyzing the cleavage of azo bonds. The optical properties of ODP exhibited excellent performance in terms of fluorescence enhancement, fluorescence lifetime (0.81 ns), and detection limit (0.86 µM) in response to SDT. Cell imaging experiments showed that ODP could effectively detect and image intracellular hypoxia and the imaging capability of ODP was studied under various conditions including cell migration, antioxidant treatment, and different incubation times. Through comprehensive in vitro and in vivo experiments, including cellular imaging and mouse tumor models, this work demonstrates the efficacy of ODP in accurately detecting and imaging hypoxia. Moreover, ODP's potential in inducing apoptosis in cancer cells offers a promising avenue for integrating diagnostic and therapeutic strategies in cancer treatment. This innovative approach not only contributes to the understanding and assessment of tumor hypoxia but also opens new possibilities for targeted cancer therapy.

Imaging Investigation of Hepatocellular Carcinoma Progress via Monitoring γ-Glutamyltranspeptidase Level with a Near-Infrared Fluorescence/Photoacoustic Bimodal Probe.

Hepatocellular carcinoma (HCC) is one of the main principal causes of cancer death, and the late definite diagnosis limits therapeutic approaches in time. The early diagnosis of HCC is essential, and the previous investigations on the biomarkers inferred that the γ-glutamyltranspeptidase (GGT) level could indicate the HCC process. Herein, a near-infrared fluorescence/photoacoustic (NIRF/PA) bimodal probe, CySO3-GGT, was developed for monitoring the GGT level and thus to image the HCC process. After the in-solution tests, the bimodal response was convinced. The various HCC processes were imaged by CySO3-GGT at the cellular level. Then, the CCl4-induced HCC (both induction and treatment) and the subcutaneous and orthotopic xenograft mice models were selected. All throughout the tests, CySO3-GGT achieved NIRF and PA bimodal imaging of the HCC process. In particular, CySO3-GGT could effectively realize 3D imaging of the HCC nodule by visualizing the boundary between the tumor and the normal tissue. The information here might offer significant guidance for the dynamic monitoring of HCC in the near future.

Mitochondrial targeted fluorescent nitrite peroxide probe for dynamic monitoring of cellular lung injury.

Herein, a mitochondrial targeted fluorescent nitrite peroxide probe CHP for dynamic monitoring of cellular lung injury was developed. For the practical delivery and selectivity, the structural features including pyridine head and borate recognition group were selected. CHP could respond to ONOO- with the 585 nm fluorescence signal. The detecting system indicated advantages such as wide linear range (0.0-30 µM), high sensitivity (LOD = 0.18 µM), high selectivity and steadiness under different environmental conditions including pH (3.0-10.0), time (48 h) and medium. In living A549 cells, the response of CHP towards ONOO- showed dose-dependent and time-dependent tendencies. The co-localization suggested that CHP could achieve mitochondrial targeting. Moreover, CHP could monitor the variation of endogenous ONOO- level and the cellular lung injury induced by LPS.

Fluorescence probe for real-time malonaldehyde detection in epilepsy model.

Oxidative stress, a condition involving an imbalance between reactive oxygen species (ROS) and antioxidants, is closely linked to epilepsy, contributing to abnormal neuronal excitability. This study introduces a novel fluorescent probe, the MDP probe, designed for the efficient detection of malondialdehyde (MDA), a critical biomarker associated with oxidative stress. The MDP probe offers several key advantages, including high sensitivity with a low detection limit of 0.08 µM for MDA, excellent selectivity for MDA even in the presence of interfering substances, and biocompatibility, making it suitable for cell-based experiments. The probe allows for real-time monitoring of MDA levels, enabling dynamic studies of oxidative stress. In vivo experiments in mice demonstrate its potential for monitoring MDA levels, particularly in epilepsy models, which could have implications for disease research and diagnosis. Overall, the MDP probe represents a promising tool for studying oxidative stress, offering sensitivity and specificity in cellular and in vivo settings. Its development opens new avenues for exploring the role of oxidative stress in various biological processes and diseases, contributing to advancements in healthcare and biomedical research.

Identification of (N-aryl-N-arylsulfonyl)aminoacetohydroxamic acids as novel urease inhibitors and the mechanism exploration.

Thirty-three (N-aryl-N-arylsulfonyl)aminoacetohydroxamic acids were synthesized in an effort to develop novel urease inhibitors. Among these compounds, 2-(N-(3-nitrophenyl)-N-(4-tert-butylphenylsulfonyl))aminoacetohydroxamic acid (e2) exhibited excellent inhibitory activity against Helicobacter pylori urease with no perceptible cytotoxicity to mammalian cells. Compound e2 showed over 690-fold higher potency than the clinical used urease inhibitor acetohydroxamic acid, reversibly inhibiting urease with a mixed mechanism. Molecular modeling revealed that (N-aryl-N-arylsulfonyl)aminoacetohydroxamic acids may possibly bind Ni ions and two hydrophobic regions with a 'Y'-like shape.

Comparative Analysis of Super-Mini Percutaneous Nephrolithotomy Combined with Flexible Ureteroscopic Lithotripsy versus Flexible Ureteroscopic Lithotripsy Alone for Treating Complex Kidney Stones: A Retrospective Study of 205 Patients.

BACKGROUND This retrospective study aimed to compare outcomes from super-mini percutaneous nephrolithotomy (SMP) combined with flexible ureteroscopic lithotripsy (FURL) and FURL alone in 205 patients with 2.5-4.2 cm diameter complex kidney stones. MATERIAL AND METHODS Between January 2018 and December 2022, 92 patients were treated with SMP combined with FURL (group A), and 113 patients were treated with FURL alone (group B). The stone-free rate (SFR), retreatment ratio, operation time, mean decline in hemoglobin level, postoperative pain visual analogue scale (VAS), and postoperative hospitalization time and complications were analyzed and compared between the 2 groups. RESULTS The SFR 3 days after the operation was 85.87% in group A, which was significantly higher than that in group B (72.57%) (P=0.021). The rate of retreatment in group A (3.26%) was significantly lower than that in group B (10.62%) (P=0.044). The SFR after 90 days was higher in group A (94.57%) than in group B (90.27%) (P=0.254). The mean decrease in hemoglobin, postoperative hospitalization duration, and VAS score 6 hours after the operation were all significantly higher in group A than in group B (P<0.05). However, there was no significant difference in operation time, VAS score at 12 and 24 hours after the operation, and complication rate. CONCLUSIONS In the treatment of complex renal stones, compared with FURL, SMP combined with FURL in the oblique supine lithotomy position has the advantages of a higher early SFR with no increased risk of complications.

Switchable metasurface for high-efficiency absorption and broadband polarization conversion based on vanadium dioxide in the terahertz frequency.

High-performance devices with superior execution will facilitate the practical application of terahertz (THz) technology and foster THz innovation. In this paper, taking advantage of the phase transition characteristics of vanadium dioxide (V O 2), a reconfigurable metasurface with absorption and polarization conversion capacities is proposed. The metallic condition of V O 2 results in the formation of a wideband absorber. It provides more than 90% absorption over a broad spectral range from 3.32 to 5.30 THz. Due to the regularity of the meta-atom, the absorber is not polarization-delicate and keeps a high retention rate in the scope of incoming angles from 0° to 45°. When V O 2 is in the insulating condition, the calculated outcomes demonstrate that the cross-polarization conversion rate can reach more than 90% in the range of 2.29-7.85 THz when x-polarized or y-polarized waves are incident vertically. The proposed metasurface is likely to be used in the fields of emitters, sensors, imaging systems, and wireless communication.

Epileptic brain fluorescent imaging reveals apigenin can relieve the myeloperoxidase-mediated oxidative stress and inhibit ferroptosis.

Myeloperoxidase (MPO)-mediated oxidative stress has been suggested to play an important role in the pathological dysfunction of epileptic brains. However, there is currently no robust brain-imaging tool to detect real-time endogenous hypochlorite (HClO) generation by MPO or a fluorescent probe for rapid high-throughput screening of antiepileptic agents that control the MPO-mediated chlorination stress. Herein, we report an efficient two-photon fluorescence probe (named HCP) for the real-time detection of endogenous HClO signals generated by MPO in the brain of kainic acid (KA)-induced epileptic mice, where HClO-dependent chlorination of quinolone fluorophore gives the enhanced fluorescence response. With this probe, we visualized directly the endogenous HClO fluxes generated by the overexpression of MPO activity in vivo and ex vivo in mouse brains with epileptic behaviors. Notably, by using HCP, we have also constructed a high-throughput screening approach to rapidly screen the potential antiepileptic agents to control MPO-mediated oxidative stress. Moreover, from this screen, we identified that the flavonoid compound apigenin can relieve the MPO-mediated oxidative stress and inhibit the ferroptosis of neuronal cells. Overall, this work provides a versatile fluorescence tool for elucidating the role of HClO generation by MPO in the pathology of epileptic seizures and for rapidly discovering additional antiepileptic agents to prevent and treat epilepsy.

Recent advances in small-molecule fluorescent probes for studying ferroptosis.

Ferroptosis is an iron-dependent, non-apoptotic form of programmed cell death driven by excessive lipid peroxidation (LPO). Mounting evidence suggests that the unique modality of cell death is involved in the development and progression of several diseases including cancer, cardiovascular diseases (CVDs), neurodegenerative disorders, etc. However, the pathogenesis and signalling pathways of ferroptosis are not fully understood, possibly due to the lack of robust tools for the highly selective and sensitive imaging of ferroptosis analytes in complex living systems. Up to now, various small-molecule fluorescent probes have been applied as promising chemosensors for studying ferroptosis through tracking the biomolecules or microenvironment-related parameters in vitro and in vivo. In this review, we comprehensively reviewed the recent development of small-molecule fluorescent probes for studying ferroptosis, with a focus on the analytes, design strategies and bioimaging applications. We also provided new insights to overcome the major challenges in this emerging field.

Multifunctional Fluorescent Probe for Simultaneously Detecting Microviscosity, Micropolarity, and Carboxylesterases and Its Application in Bioimaging.

Based on OR logic gate, we proposed a smart near-infrared (NIR) fluorescent probe, named VPCPP, for simultaneously monitoring local microviscosity, micropolarity, and carboxylesterases (CEs) in living cells through blue and red channels. This proposed probe was capable of distinguishing cancer cells from normal cells and had good potential for identifying living liver cell lines. Furthermore, the fluctuations of the three analytes of interest in different cell status was successfully explored. Particularly, facilitated with high-content analysis (HCA) and VPCPP, a simple and efficient high-throughput screening (HTS) platform was first constructed for screening antitumor drugs and studying their effect on the analytes. For the first time, we found that sorafenib-induced ferroptosis led to an increase in the microviscosity and up-regulation of CEs at the same time. Additionally, the procedure that aristolochic acid (AA) induced the overexpression of CEs was verified. Besides, VPCPP was utilized for imaging the variations of the two microenvironment parameters and CEs in the inflammation model. Finally, VPCPP was able to image the tumor ex vivo and in vivo through two channels and one channel separately, as well as to visualize the kidneys and liver ex vivo with dual emissions, which indicated that the probe had great potential for imaging applications such as medical diagnosis, preclinical research, and imaging-guided surgery.

Metasurface absorber with ultra-thin thickness designed for a terahertz focal plane array detector.

Terahertz (THz) refers to electromagnetic waves with frequency from 0.1 to 10 THz, which lies between millimeter waves and infrared light. This paper proposes an ultra-thin metasurface absorber which is perfectly suited to be the signal coupling part of terahertz focal plane array (FPA) detector. The absorptance of the proposed metasurface is higher than 80% from 4.46 to 5.76 THz (25.4%) while the thickness is merely 1.12 µm (0.018 λ). Since the metasurface absorber will be applied to terahertz FPA detector which requires planar array formation, it is divided into meta-atoms. Each meta-atom consists of the same unit cell layout, and air gaps are introduced between adjacent meta-atoms to enhance the thermal isolation, which is crucial for FPA detector to obtain desired imaging results. Due to the symmetrical layout of meta-atoms, absorptance keeps stable for different polarized waves, moreover, good absorptance could also be achieved for incidence angles range of ± 30 °. Spectral measurements show good agreement with the simulation. As a result, features of ultra-thin thickness, polarization insensitivity, and high absorptance make the proposed metasurface absorber well suited to highly efficient coupling of terahertz signals in FPA detector.