Coordination Synergistic-Induced J-Aggregation Enhanced Fluorescent Performance of HBT-Excimers and Imaging Applications.

Developing a novel strategy to improve the optical performances of fluorescent probes is a vital factor in elevating its practical application; viz., novel biocompatible fluorescent probes with excellent multifunctions exhibited unparalleled advantages in probing functions of intracellular molecules to elucidate intracellular events in living systems. Herein, we have successfully constructed a new strategy that aggregation and coordination synergistically induce (2-hydroxylphenyl-benzothiazole) HBT derivatives to form excimers with large red-shifted fluorescence and application for insight into stress-response zinc fluctuations in living systems. We have synthesized four HBT-based derivatives and deeply investigated the response mechanism by fluorescent spectral studies, demonstrating that probes 3 and 4 showcased large red shifts in emission wavelength due to J-aggregation. More interestingly, the fluorescence of probe 4 was significantly enhanced in the presence of a zinc ion, suggesting that zinc coordination synergistically induced J-aggregation. Probe 4 was successfully applied to image zinc fluctuations in different models of living systems, proving that this probe is a powerful tool to unveil the relationship between invasive stress and diseases by monitoring endogenous zinc fluctuations.

Transcriptional regulation of cellobiose utilization by PRD-domain containing Sigma54-dependent transcriptional activator (CelR) and catabolite control protein A (CcpA) in Bacillus thuringiensis.

Cellobiose, a ß-1,4-linked glucose dimer, is a major cellodextrin resulting from the enzymatic hydrolysis of cellulose. It is a major source of carbon for soil bacteria. In bacteria, the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS), encoded by the cel operon, is responsible for the transport and utilization of cellobiose. In this study, we analyzed the transcription and regulation of the cel operon in Bacillus thuringiensis (Bt). The cel operon is composed of five genes forming one transcription unit. ß-Galactosidase assays revealed that cel operon transcription is induced by cellobiose, controlled by Sigma54, and positively regulated by CelR. The HTH-AAA+ domain of CelR recognized and specifically bound to three possible binding sites in the celA promoter region. CelR contains two PTS regulation domains (PRD1 and PRD2), which are separated by two PTS-like domains-the mannose transporter enzyme IIA component domain (EIIAMan) and the galactitol transporter enzyme IIB component domain (EIIBGat). Mutations of His-546 on the EIIAMan domain and Cys-682 on the EIIBGat domain resulted in decreased transcription of the cel operon, and mutations of His-839 on PRD2 increased transcription of the cel operon. Glucose repressed the transcription of the cel operon and catabolite control protein A (CcpA) positively regulated this process by binding the cel promoter. In the celABCDE and celR mutants, PTS activities were decreased, and cellobiose utilization was abolished, suggesting that the cel operon is essential for cellobiose utilization. Bt has been widely used as a biological pesticide. The metabolic properties of Bt are critical for fermentation. Nutrient utilization is also essential for the environmental adaptation of Bt. Glucose is the preferred energy source for many bacteria, and the presence of the phosphotransferase system allows bacteria to utilize other sugars in addition to glucose. Cellobiose utilization pathways have been of particular interest owing to their potential for developing alternative energy sources for bacteria. The data presented in this study improve our understanding of the transcription patterns of cel gene clusters. This will further help us to better understand how cellobiose is utilized for bacterial growth.

Unraveling the complex dynamics of signaling molecules in cellular signal transduction.

Signaling molecules in cellular responses to foreign stimuli are described as static up- or down-concentration changes during signal transduction. This is because analytical methods for transducing molecules are much slower than the signaling events. In this study, we develop a dynamic cell model and reveal the temporal regulation of signal transduction events in response to reactive oxygen species (ROS). The model contained a set of 10 batches of redox-modified cells that mimic the temporal ROS accumulation events. Validating this dynamic cell model, we discover that cells survive early ROS attacks by activating the Nrf2/polysulfide/p62/CDK1 pathway. Nearly all signaling molecules exhibit time-dependent V-shape or inverse V-shape activation/feedback regulation dynamics in response to ROS accumulation. The results show that the dynamic cell model approach is invaluable for revealing complex signal intensity- and time-dependent cell signaling events.

Waste iron scraps promote anammox bacteria to resist inorganic carbon limitation.

The anaerobic ammonium oxidation (anammox) process is adversely affected by the limitation of inorganic carbon (IC). In this research, a new technique was introduced to assist anammox biomass in counteracting the adverse effects of IC limitation by incorporating waste iron scraps (WIS), a cheap and easily accessible byproduct of lathe cutting. Results demonstrated that reducing the influent IC/TN ratio from 0.08-0.09 to 0.04 resulted in a 20 % decrease in the nitrogen removal rate (NRR) for the control reactor, with an average specific anammox activity (SAA) of 0.65 g N/g VSS/day. Nevertheless, the performance of the WIS-assisted anammox reactor remained robust despite the reduction in IC supply. In fact, the NRR and SAA of the WIS-assisted reactor exhibited substantial improvements, reaching approximately 1.86 kg/(m3·day) and 0.98 g N/g VSS/day, respectively. These values surpassed those achieved by the control reactor by approximately 39 % and 51 %, respectively. The microbial analysis confirmed that the WIS addition significantly stimulated the proliferation of anammox bacteria (dominated by Candidatus Kuenenia) under IC limitation. The anammox gene abundances in the WIS-assisted anammox reactor were 3-4 times higher than those in the control reactor. Functional genes prediction based on the KEGG database revealed that the addition of WIS significantly enhanced the relative abundances of genes associated with nitrogen metabolism, IC fixation, and central carbon metabolism. Together, the results suggested that WIS promoted carbon dioxide fixation of anammox species to resist IC limitation. This study provided a promising approach for effectively treating high ammonium-strength wastewater using anammox under IC limitation.

Superselective renal arterial embolization for severe postpercutaneous nephrolithotomy haemorrhage: clinical characteristics and risk factors for initial failure.

OBJECTIVE: To identify the clinical characteristics of patients who underwent superselective renal arterial embolization (SRAE) after percutaneous nephrolithotomy (PCNL) and to explore the risk factors for failed initial SRAE after PCNL. MATERIALS AND METHODS: Patients who underwent SRAE for severe haemorrhage following PCNL between January 2014 and December 2020 were included in the study. The clinical data of those patients and the parameters and characteristics of the perioperative PCNL and SRAE procedures were collected and analysed. RESULTS: A total of 243 patients were included in this study. A total of 139 patients (57.2%) had a pseudoaneurysm, 25 (10.3%) had an arteriovenous fistula, 50 (20.6%) patients had both a pseudoaneurysm and an arteriovenous fistula, and 29 (11.9%) had an arterial laceration. In 177 patients with single percutaneous access, 125 (70.6%) patients exhibited nontract haemorrhage, and 55 (31.1%) patients exhibited multiple bleeding sites. In 66 patients with multiple percutaneous access, 44 (66.7%) patients exhibited nontract haemorrhage, and 32 (48.5%) patients exhibited multiple bleeding sites. The decrease in Hb before SRAE was 41.4 ± 19.8 g/L. The mean time between PCNL surgery and initial SRAE was 6.4 ± 4.9 days. Serum creatinine was increased after the SRAE procedure. Initial SRAE was successful in 229 (94.2%) patients and failed in 14 (5.8%) patients. Multivariate regression demonstrated that hydronephrosis < 20 mm, total ultrasonographic guidance, solitary kidney, previous ipsilateral renal surgery, PCNL duration > 90 min and multiple bleeding sites were potential risk factors for initial embolization failure. CONCLUSION: Percutaneous access was not the most important reason for post-PCNL severe haemorrhage. SRAE is effective for the treatment of severe haemorrhage following PCNL; however, several factors have an impact on the success of initial SRAE. Additionally, the SRAE procedure may affect renal function.

Fluorescent imaging to provide visualized evidences for mercury induced hypoxia stress.

As one typical toxic and dangerous heavy metal, mercury brings incalculable hazards to the environment and human, the mechanism at the molecular level is unclear. There is no visualized evidence to support directly that mercury ions (Hg2+) exposure may induce secondary stress, which is associated with the risk of hypoxia microenvironment in biological systems. Hypoxia occurs in many physiological and pathophysiological processes in the living system, accompanying overexpression of various biomarkers, such as nitroreductase (NTR). Hence, we had successfully developed two NTR-selective fluorescent probes with excellent performance for evaluating the hypoxia degree in vivo and in vitro. We visualized and qualitatively monitored the fluctuations of the endogenous NTR levels in living cells and zebrafish. The imaging results exhibited that different doses of Hg2+ exposure elevated the NTR levels and the same trend in changes of NTR as extrinsic hypoxia exposure, suggesting that Hg2+ exposure induced microenvironmental changes resulting in the hypoxia stress. This is the first time to provide visual evidence to support that Hg2+ stress may involve in the intracellular hypoxia microenvironment through monitoring the dynamic of NTR levels in the living systems. Our results may provide a novel insight into the molecular mechanisms of typical heavy metal element induced toxicity.

A small molecule fluorescent probe for mercury ion analysis in broad low pH range: Spectral, optical mechanism and application studies.

Development of new fluorescent probes for mercury ion analysis in environmental or living organism is undergoing quick growth due to its detrimental toxicity to environmental safety, ecological security, and human being. However, in most cases, the industrial waste water is acidic whereas it remains a great challenge to real-time monitor mercury ion directly at low pH using small molecule fluorescence probe. In this study, we have successfully designed and synthesized the Naph (1, 8-Naphthalimide derivative) -based small molecule probe termed as Naph-NSS capable of monitoring mercury ion in a broad range at low pH (from 2.0 to 7.0). The solid spectral studies demonstrated the high sensitivity and selectivity of the probe towards mercury ion among various species. After binding with Hg2+, the fluorescence of Naph-NSS greatly enhanced, and the mechanism of which was investigated by DFT studies. The probe was able to be loaded on paper strip for instant and fast detection of mercury ions. In addition, the probe is also suitable for detection of mercury ion in environmental samples, living cells and in vivo.

Design and Synthesis of Neutralizable Fondaparinux.

Fondaparinux, a clinically approved anticoagulant pentasaccharide for the treatment of thrombotic diseases, displays better efficacy and biosafety than other heparin-based anticoagulant drugs. However, there is no suitable antidote available for fondaparinux to efficiently manage its potential bleeding risks, thereby precluding its widespread use. Herein, we describe a convergent and stereocontrolled approach to efficiently synthesize an aminopentyl-functionalized pentasaccharide, which is further used to prepare fondaparinux-based biotin conjugates and clusters. Biological activity evaluation demonstrates that the anticoagulant activity of the fondaparinux-based biotin conjugate and trimer is, respectively, neutralized by avidin and protamine as effective antidotes. This work suggests that our synthetic biotin conjugate and trimer have potential for the development of neutralizable and safe anticoagulant drugs.

Insight into sulfur dioxide and its derivatives metabolism in living system with visualized evidences via ultra-sensitive fluorescent probe.

Sulfur dioxide (SO2) and its derivatives have long been considered as hazardous environmental pollutants but commonly used as food additives in safe dose range. They also could be produced from biological metabolism process of sulfur-containing amino acids. However, their physiological roles remain extremely obscure mainly due to lack of efficient tools for monitoring and imaging strategy establishment. Furthermore, most of current studies of this aspect focus on novel probe design or just imaging them rather than on the ins and outs. Therefore, there is a high significance of establishing highly sensitive detection strategy for monitoring SO2 derivatives in living systems, food and environment. Herein, we design a fluorescent probe MS-Bindol for sensitively detecting SO2 derivatives with a low detection limit (0.2 nM). We have established an imaging strategy for investigation of SO2 derivatives metabolism in living cells and zebrafish, providing visualize evidences and verified that SO2 derivatives could be synthetized from thiosulfate and glutathione(GSH) and be hardly consumed by using sulfite oxidase inhibitors (ferricyanide or arsenite). Moreover, the probe also exhibits excellent practicability in food as well as environmental samples. Our studies may help biologist for better understanding SO2 derivatives metabolism and deeply explore their physiological roles in biological systems.

Visualizing and evaluating mitochondrial cysteine via near-infrared fluorescence imaging in cells, tissues and in vivo under hypoxia/reperfusion stress.

Increasingly grim environmental pollutions are closely related with the occurrence and development of diseases. However, it's obscure how environmental stress disturbs the normal physiological process, and then how endogenous reactive species mend the cases. Hypoxia/reperfusion (H/R), a common and intractable injury in aquaculture and clinic, can induce oxidative stress and ultimately cause irreversible injury to organism. Cysteine (Cys) plays essential roles in maintaining transduction of numerous reactive species and redox homeostasis in subcellular structures, cells and organisms. A great deal of fluorescence research about Cys are focusing on development of selective probes but with poor exploration of the biofunction under environmental stress. Therefore, it is of great significance to examine the bio-effects of Cys against H/R stress. In the present work, we design a fluorescent probe BCy-AC for in situ detecting Cys, the unique Enol-Keto tautomerization of fluorophore BCy-Keto propels the reaction process which will improve the sensitivity and potential application performance of the probe. BCy-AC is conveniently applied to visualize Cys in HT-22 cells, zebrafish and mice tissues. Moreover, imaging results obtained from H/R models reveal that endogenous Cys changes with hypoxia and reperfusion time and Cys pretreatment effectively defend H/R injury in cells and in vivo.

Evaluation of cyclooxygenase-2 fluctuation via a near-infrared fluorescent probe in idiopathic pulmonary fibrosis cell and mice models.

Idiopathic pulmonary fibrosis (IPF) is a devastating and fatal interstitial lung disease due to various challenges in diagnosis and treatment. Due to its complicated pathogenesis and difficulty in early diagnosis, there is no effective cure. Cyclooxygenase-2 (COX-2) is inextricably associated with pulmonary fibrosis. The abnormal level of COX-2 leads to extremely exacerbated pulmonary fibrosis. Therefore, we reported a near-infrared fluorescent probe Cy-COX to detect the fluctuation of COX-2 levels during pulmonary fibrosis and explain its important protective effect. The probe Cy-COX showed a significant enhancement of fluorescence signal to COX-2 with excellent selectivity and sensitivity. In order to clarify the relationship between COX-2 and pulmonary fibrosis, we used the probe Cy-COX to detect COX-2 fluctuation in organisms with pulmonary fibrosis. The results showed that the COX-2 level increased in the early stage and decreased in the late stage with the aggravation of pulmonary fibrosis. Furthermore, up-regulation of COX-2 levels can effectively alleviate the severity of pulmonary fibrosis. Therefore, Cy-COX is a fast and convenient imaging tool with great potential to predict the early stage of pulmonary fibrosis and evaluate the therapeutic effects.

A cysteine-selective fluorescent probe for monitoring stress response cysteine fluctuations.

Rare studies provided evidence for the real-time monitoring of stress response cysteine fluctuations. Here, we have successfully designed and synthesized a cysteine-selective fluorescent probe 1 to monitor stress response Cys fluctuations, providing visual evidence of Hg2+ regulated cysteine fluctuations for the first time, which may open a new way to help researchers to reveal the mechanism of heavy metal ion poisoning.

A chemical covalent tactic for bio-thiol sensing and protein labeling agent design.

Discovering novel chemical reactions is important for bioanalysis. Herein, we report a tactic for bio-thiol sensing and protein labeling agent design by the installation of a sulfoxide group onto the skeleton of various fluorophores, and powerfully validate its abilities, which may shed light on the development of specific protein tags to give insight into their biological functions.

A reaction-based ratiometric fluorescent probe for mercury ion detection in aqueous solution.

Mercury ions are crucially harmful to ecosystem and human being due to their characters of bioaccumulation and difficulty of biochemical degradation. Therefore, development of mercury ion detection methods has attracted increasing interests recently. In this study, we successfully synthesized a hydroxyphenylbenzothiazole (HBT)-based fluorescent probe HBT-Hg in an extremely simple manner for mercuric ions detection. The spectral studies revealed that the probe HBT-Hg could react with Hg2+ selectively and sensitively in PBS buffer (10 mM, pH = 7.40), showing ratiometric fluorescent changes from blue to light green. The response mechanism of the probe HBT-Hg and Hg2+ was finally confirmed by HPLC analysis, viz., the probe HBT-Hg converted to its precursor compound 1. Finally, the probe HBT-Hg was successfully applied in monitoring Hg2+ in living A549 cells.

A near-infrared fluorescent probe for observing thionitrous acid-mediated hydrogen polysulfides formation and fluctuation in cells and in vivo under hypoxia stress.

Hydrogen polysulfides (H2Sn, n>1) as important intracellular reactive sulfur species (RSS) are believe to be responsible for cellular redox regulation. Lots of researches about H2Sn focusing on their formation, detection and bio-function in signalling regulation are spring up but with poor understanding, especially for biosynthesis and bio-function remain complicated and confusing. Recent studies reveal that thionitrous acid (HSNO) as potential intermediate linked signalling molecules of nitrogenous and sulphureous during biotic redox regulation. However, there are limited evidences for supporting the interrelation and bioeffect between HSNO and H2Sn. Herein, we have successfully designed a near-infrared (NIR) fluorescent probe ((2-fluoro-5-nitrobenzoyl)oxy)-Benzo[e]cyanine (BCy-FN) for detection H2Sn and for the first time observing HSNO-mediated H2Sn generation in cells and in vivo. The probe is harvested from fluorophore BCy-Keto and 2-fluoro-5-nitrobenzoic acid in one step, featuring mitochondria localization. The unique Enol-Keto tautomerization of fluorophore enables the probe becomes more sensitive and has powerful application. Hypoxia model has been constructed and powerfully interpreted the pretreatment of HSNO for zebrafish hypoxia process effectively improves H2Sn levels and defends the hypoxia induced brain damage. We believe the present studies will help environmentalist and biologist for better understanding of biosynthesis and bio-function in HSNO-mediated H2Sn formation process under hypoxia stress.

Glutathione Peroxidase-Activatable Two-Photon Ratiometric Fluorescent Probe for Redox Mechanism Research in Aging and Mercury Exposure Mice Models.

Solid evidence confirms that glutathione peroxidase (GPx) is a kind of vital protease in the first-line antioxidant defense system and participates in regulation of redox homeostasis as well as the pentose phosphate pathway. However, the current methods cannot achieve real-time and in situ visualization studies of GPx. In addition, GPx is highly reactive and susceptible to external interference, and there is rare research for exploring the roles of GPx under environmental factor exposure. Herein, we report a novel two-photon ratiometric fluorescent probe (TP-SS) for GPx detection for the first time. Using TP-SS, we explore the reversible catalytic cycle and the antioxidant mechanisms of GPx/GSH redox pool in aging and mercury exposure models. We detect the concentration fluctuation of GPx in aging and mercury exposure mice models. Also, we perform GPx detection in deep brain tissue and the imaging depth up to 100 µm. We believe that the novel two-photon ratiometric fluorescent probe TP-SS can facilitate the development of GPx-targeting tools and offer great advances in exploring the physiological/pathological functions of GPx.

Evaluating the Protective Effects of Mitochondrial Glutathione on Cerebral Ischemia/Reperfusion Injury via Near-Infrared Fluorescence Imaging.

Cerebral ischemia/reperfusion (I/R) is common and intractable in the clinic, associated with the outburst of reactive oxygen species (ROS) in mitochondria. Although numerous research studies have been conducted to prove the protective-effect roles of glutathione (GSH) in this event, the changes in GSH concentrations in living cells remain largely unexplained, and there is scarce evidence by fluorescence imaging for its roles. Herein we have designed and synthesized two distinctive "off-on" near-infrared (NIR) fluorescent probes BCy-SeSe and BCy-SS based on a new fluorophore BCy-Keto for specific response to mitochondrial GSH changes during the cerebral I/R process. Both of them exhibit powerful targeting capability in mitochondria and excellent photophysical properties toward endogenous GSH with high selectivity and sensitivity. In contrast to BCy-SS, BCy-SeSe was screened for biological application on account of its faster response rate. We have utilized BCy-SeSe to real-time image GSH during the cerebral I/R process in living cells and the mice focal cerebral ischemia model (middle cerebral artery occlusion, MCAO), and these intensive studies revealed that low GSH levels were associated with aggravation of apoptosis and cerebral infarction. Pretreatment with GSH synthase inhibitor aggravates damage while GSH-ester alleviates damage, confirming that GSH is effective on the cerebrum for protection from I/R. All the results demonstrated that the probes were powerful tools for investigating mitochondrial GSH during the I/R process in living cells and in vivo.

High Spatiotemporal Resolution Observation of Glutathione Hydropersulfides in Living Cells and Tissue via a Two-Photon Ratiometric Fluorescent Probe.

Glutathione hydropersulfides (GSSH) are alluded to play crucial roles in signal transduction, redox homeostasis, and metabolic regulation. However, the detailed biological functions of GSSH in these aspects are extremely ambiguous. The key barrier to understand the role of GSSH in biological systems is a lack of detection tools with high spatiotemporal resolution. To address the issues, we are seeking novel chemical tools for GSSH detection. We herein develop the first two-photon ratiometric fluorescent probe (TP-Dise) for GSSH detection with high spatial and temporal resolution in living cells and tissue. On the basis of our probe TP-Dise, we investigate the biosynthesis of GSSH, and the results indicate that GSSH is mainly from two sulfurtransferases, CBS and CSE. Furthermore, we explore the biological function of GSSH in protecting cells from mercury ion-induced cell damage for the first time. The experimental results indicate that mercury ions may induce cell death by causing mitochondrial autophagy. GSSH acts both as antagonist and as antioxidant and can effectively alleviate the damage caused by mercury stress.

A near-infrared fluorescent probe for evaluating endogenous hydrogen peroxide during ischemia/reperfusion injury.

Hydrogen peroxide (H2O2), as a major component of reactive oxygen species (ROS), plays an important role in normal physiological processes. A H2O2 burst also occurs in the ischemia/reperfusion (I/R) process and causes a series of physiological and pathological injuries. Therefore, it is important to determine concentration fluctuations of H2O2. Here we develop a ratiometric fluorescent probe, Cy-ArB, which shows high selectivity and sensitivity toward H2O2. The fluorescence response of the probe is triggered by the reaction of borate esters with H2O2, and this process releases a near-infrared heptamethine cyanine fluorophore which has the ability of mitochondrial tracing. Hence, the probe can be used for real-time monitoring of H2O2 fluctuations in the mitochondrial respiration chain. Finally, we explore the fluctuations of H2O2 in cells and in vivo during the I/R process using the probe Cy-ArB. The results of our experiments prove that our probe is a potential candidate for clinical surgery pre-evaluation.

A ratiometric fluorescent probe of methionine sulfoxide reductase with an improved response rate and emission wavelength.

A ratiometric fluorescent probe of methionine sulfoxide reductase, Msr-Ratio, was disclosed for monitoring the enzyme activity in vitro and in live cells. The probe displayed favorable properties such as a nearly 400-fold fluorescence change, fast response rate (<30 min), large Stokes shift (120 nm), and green emission (550 nm).