A Novel Near-Infrared Fluorescent Probe for the Detection of Endogenous Peroxynitrite (ONOO-) in Living Cells.

In this study, we present a novel near-infrared (NIR) fluorescent probe Nile-ONO designed for the selective and sensitive detection of ONOO-. The probe Nile-ONO employed Nile red as the fluorophore, with diphenylphosphinate serving as the reaction site. In the presence of ONOO-, the probe Nile-ONO exhibits remarkable fluorescence enhancement at 659 nm, with a response time of less than 20 min and a low detection limit of 0.32 µM. Importantly, MTT assays demonstrate low cytotoxicity in living cells. Furthermore, Nile-ONO has excellent imaging capabilities for endogenous ONOO-. Overall, this work introduces a valuable new method for the rapid detection of ONOO- in biological systems.

Electrochemiluminescence-Microscopy for microRNA Imaging in Single Cancer Cell Combined with Chemotherapy-Photothermal Therapy.

In this work, a new technology using ECL as a microscopy to parallel image miRNA-21 in single cancer cell was built. Phorbol-12-myristate-13-acetate (PMA) loaded gold nanocages (Au NCs) was closed with DNA gate which could be recognized and opened by miRNA-21 in HeLa cell. PMA was then released and further induced HeLa cells to produce reactive oxygen species (ROS; including O2-•, •OH and H2O2 etc.). With H2O2 as coreactant and luminol as ECL active material, ECL imaging of intracellular miRNA-21 in single HeLa cell was obtained by EMCCD. Moreover, ROS therapy and photothermal therapy (PPT) of Au NCs@PMA probe were also motivated by in situ miRNA-21 marker instead of the external source. The combined therapy leads to dramatically enhanced ability for cancer cell killing. Au NCs@PMA probe alone could not only achieve a high sensitivity and high resolution ECL-microscopy for imaging of intracellular miRNA-21 for the first time, but also realize the integrated diagnosis like ROS induced tumor damage and photothermal-induced intelligent therapy. This multifunctional platform is believed to be capable of playing an important role in future oncotherapy by the synergistic effects between chemotherapy and photothermal therapy.

Triosephosphate isomerase tyrosine nitration induced by heme-NaNO2 -H2 O2 or peroxynitrite: Effects of different natural phenolic compounds.

Peroxynitrite and heme peroxidases (or heme)-H2 O2 -NaNO2 system are the two common ways to cause protein tyrosine nitration in vitro, but the effects of antioxidants on reducing these two pathways-induced protein nitration and oxidation are controversial. Both nitrating systems can dose-dependently induce triosephosphate isomerase (TIM) nitration, however, heme-H2 O2 -NaNO2 was less destructive to protein secondary structures and led to more nitrated tyrosine residue than 3-morpholinosydnonimine hydrochloride (SIN-1, a peroxynitrite donor). Both of desferrioxamine and catechin could inhibit TIM nitration induced by heme-H2 O2 -NaNO2 and SIN-1 and protein oxidation induced by SIN-1, but promoted heme-H2 O2 -NaNO2 -induced protein oxidation. Moreover, the antagonism of natural phenolic compounds on SIN-1-induced tyrosine nitration was consistent with their radical scavenging ability, but no similar consensus was found in heme-H2 O2 -NaNO2 -induced nitration. Our results indicated that peroxynitrite and heme-H2 O2 -NaNO2 -induced protein nitration was different, and the later one could be a better model for anti-nitration compounds screening.

A novel lysosome-targeted fluorescent probe for precise formaldehyde detection in water samples, living cells and breast cancer tumors.

This study investigated the potential ability of the fluorescent probe Ly-CHO to detect formaldehyde (FA) in living cells and tumor-bearing mice. Ly-CHO exhibited great selectivity, excellent sensitivity, and rapid response to FA, making it a valuable tool for tracking FA concentration changes. The probe was also found to target lysosomes specifically. Furthermore, Ly-CHO showed an obvious fluorescence increase in endogenous CHO detection after adding tetrahydrogen folic acid (THFA). This study validated Ly-CHO's possibility for FA imaging in vivo, with potential applications in understanding formaldehyde-related diseases.

Electrochemiluminescence Biosensor for Nucleolin Imaging in a Single Tumor Cell Combined with Synergetic Therapy of Tumor.

Nucleolin, a nuclear biological multifunctional protein, plays significant roles in modulating the proliferation, survival, and apoptosis of tumor cells. Different from the traditional electrochemiluminescence (ECL) method, a new ECL biosensor was built to perform ECL imaging of nucleolin in a single HeLa cell with high sensitivity and throughput. Briefly, mesoporous silica nanoparticles (MSN) loaded with doxorubicin (DOX) and phorbol 12-myristate 13-acetate (PMA) were used as drug carriers and could be specifically opened by nucleolin in a HeLa cell. PMA then induced the HeLa cell to produce reactive oxygen species (ROS) and realized ECL imaging of nucleolin. After that, ROS could damage DNA and proteins of the tumor cell and DOX could induce the apoptosis of HeLa cells by inhibiting genetic material, nucleic acid, synthesis. HeLa cells were then efficiently killed by DOX and ROS in a synergetic pathway. Herein, a new ECL biosensor for ECL imaging of nucleolin in a single HeLa cell and synergetic tumor therapy was built.

Peroxynitrite scavenger FeTPPS effectively inhibits hIAPP aggregation and protects against amyloid induced cytotoxicity.

Type 2 diabetes (T2D) is associated with pancreatic ß-cell dysfunction, which can be induced by oxidative stress or/and the aggregation of human islet amyloid polypeptide (hIAPP). Therefore, ONOO- and hIAPP become the crucial targets of T2D treatment. Previously, we found heme could be an effective inhibitor of hIAPP aggregation. However, heme causes serious toxic effects on cells, tissues and organs through oxidative stress, which block it as a potential drug candidate for T2D treatment. 5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrinato iron(III) chloride (FeTPPS), a water-soluble derivative of heme, is recognized as a high-efficient ONOO- decomposition catalyst, which is reported to have a great therapeutic potential in ONOO- -related diseases, including T2D. Here, we explored the potentiality of FeTPPS to be an inhibitor of hIAPP aggregation and the protective effects on cytotoxicity of hIAPP aggregation. It was found that the interaction between FeTPPS and hIAPP remarkably affected hIAPP fibrillation by both stabilizing hIAPP monomers and disaggregating the long fibrils into small oligomeric species. Furthermore, unlike heme, the addition of FeTPPS completely reversed the cytotoxicity and ROS level induced by hIAPP, which was consistent with its strong inhibitory activity. These results implied that FeTPPS could be a promising agent for the treatment of T2D.

Dual Anti-/Prooxidant Behaviors of Flavonoids Pertaining to Cu(II)-Catalyzed Tyrosine Nitration of the Insulin Receptor Kinase Domain in an Antidiabetic Study.

Flavonoid, as a potent antioxidant, exerts many beneficial effects in type 2 diabetes, whereas the prooxidative property may be also important in vivo if copper is involved. Here, we chose an insulin receptor kinase domain fragment (KK-1, residues 1126-1165), containing the A-loop of the receptor as well as three key autophosphorylation sites (Tyr1158, Tyr1162, and Tyr1163) associated with receptor signal transduction to investigate the roles and the structure-activity relationship of three antidiabetic flavonoids (kaempferol, luteolin, and apigenin) and two others with a similar structure (diosmetin and genistein), on modulation of Cu(II)-mediated tyrosine nitration and the corresponding effect on its functional phosphorylation in the Cu2+/H2O2/NO2- system. We found that both properties of flavonoid played roles on inhibition of Cu(II)-mediated protein nitration in the H2O2/NO2- system: (1) on the one hand, flavonoid scavenged free radicals as antioxidants, inhibited tyrosine nitration, and thus inhibited the reduction of tyrosine phosphorylation caused by tyrosine nitration; and (2) on the other hand, flavonoid promoted •OH production as a prooxidant, which increased 3,3'-dityrosine formation. The formation of 3,3'-dityrosine decreased Cu2+-induced tyrosine nitration and thus interfered with its phosphorylation. This study confirms that the weight relationship between antioxidation and prooxidation of a flavonoid needs to be studied clearly before nutritional and medical applications.

Nitration of amyloid-ß peptide (1-42) as a protective mechanism for the amyloid-ß peptide (1-42) against copper ion toxicity.

It is known that copper ion (Cu(II)) binds to amyloid-ß peptide (Aß), induces Aß oligomer formation and ultimately exacerbates Aß-aggregation neurotoxicity in Alzheimer's disease (AD). It becomes interesting to know that how this chemical modification of Aß would affect interaction of Aß and Cu(II) and their roles in the development of AD. In this work, we investigated the interaction of Aß1-42 nitration with the toxic Cu(II). It showed that Cu(II)induced Aß1-42 nitration in the presence of nitrite and hydrogen peroxide. Circular dichroism studies also revealed significant conformational change of Aß1-42 and Tyr10 nitrated amyloid-ß peptide(1-42) (Aß1-42NT) when interacting with Cu(II). Even though nitration did not alter the binding of Aß1-42 to Cu(II) or the peroxidative activity of Aß1-42-Cu(II) complex, nitration ameliorated the aggregation and neurotoxicity of Aß1-42 induced by Cu(II), which was also further confirmed by the cell study. Given our previous findings that Aß nitration dramatically inhibited its aggregation and thus reduced its toxicity, we speculated that nitration of Aß1-42 altered its intermolecular interaction, which protected itself against the toxicity of Cu(II). Based on this hypothesis, we propose that nitration of Aß1-42 may be an important protective mechanism for normal function of Aß1-42 and deserves more attention in AD drug development.

Synergistic Interaction of Light Alcohol Administration in the Presence of Mild Iron Overload in a Mouse Model of Liver Injury: Involvement of Triosephosphate Isomerase Nitration and Inactivation.

It is well known that iron overload promotes alcoholic liver injury, but the doses of iron or alcohol used in studies are usually able to induce liver injury independently. Little attention has been paid to the coexistence of low alcohol consumption and mild iron overload when either of them is insufficient to cause obvious liver damage, although this situation is very common among some people. We studied the interactive effects and the underlining mechanism of mild doses of iron and alcohol on liver injury in a mouse model. Forty eight male Kunming mice were randomly divided into four groups: control, iron (300 mg/kg iron dextran, i.p.), alcohol (2 g/kg/day ethanol for four weeks i.g.), and iron plus alcohol group. After 4 weeks of treatment, mice were sacrificed and blood and livers were collected for biochemical analysis. Protein nitration level in liver tissue was determined by immunoprecipitation and Western blot analysis. Although neither iron overload nor alcohol consumption at our tested doses can cause severe liver injury, it was found that co-administration of the same doses of alcohol and iron resulted in liver injury and hepatic dysfunction, accompanied with elevated ratio of NADH/NAD+, reduced antioxidant ability, increased oxidative stress, and subsequent elevated protein nitration level. Further study revealed that triosephosphate isomerase, an important glycolytic enzyme, was one of the targets to be oxidized and nitrated, which was responsible for its inactivation. These data indicate that even under low alcohol intake, a certain amount of iron overload can cause significant liver oxidative damage, and the modification of triosephosphate isomerasemight be the important underlining mechanism of hepatic dysfunction.

Iron increases diabetes-induced kidney injury and oxidative stress in rats.

Diabetic nephropathy is both a common and a severe complication of diabetes mellitus. Iron is an essential trace element. However, excess iron is toxic, playing a role in the pathogenesis of diabetic nephropathy. The present study aimed to determine the extent of the interaction between iron and type 2 diabetes in the kidney. Male rats were randomly assigned into four groups: control, iron (300-mg/kg iron dextran), diabetes (a single dose of intraperitoneal streptozotocin), and iron + diabetes group. Iron supplementation resulted in a higher liver iron content, and diabetic rats showed higher serum glucose compared with control rats, which confirmed the model as iron overload and diabetic. It was found that iron + diabetes group showed a greater degree of kidney pathological changes, a remarkable reduction in body weight, and a significant increase in relative kidney weight and iron accumulation in rat kidneys compared with iron or diabetes group. Moreover, malondialdehyde values in the kidney were higher in iron + diabetes group than in iron or diabetes group, sulfhydryl concentration and glutathione peroxidase activity were decreased by the diabetes and iron + diabetes groups, and protein oxidation and nitration levels were higher in the kidney of iron + diabetes group as compared to iron or diabetes group. However, iron supplementation did not elevate the glucose level of a diabetic further. These results suggested that iron increased the diabetic renal injury probably through increased oxidative/nitrative stress and reduced antioxidant capacity instead of promoting a rise in blood sugar levels; iron might be a potential cofactor of diabetic nephropathy, and strict control of iron would be important under diabetic state.