Broadly Applicable Strategy for the Fluorescence Based Detection and Differentiation of Glutathione and Cysteine/Homocysteine: Demonstration in Vitro and in Vivo

Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are small biomolecular thiols that are present in all cells and extracellular fluids of healthy mammals. It is well-known that each plays a separate, critically important role in human physiology and that abnormal levels of each are predictive of a variety of different disease states. Although a number of fluorescence-based methods have been developed that can detect biomolecules that contain sulfhydryl moieties, few are able to differentiate between GSH and Cys/Hcy. In this report, we demonstrate a broadly applicable approach for the design of fluorescent probes that can achieve this goal. The strategy we employ is to conjugate a fluorescence-quenching 7-nitro-2,1,3-benzoxadiazole (<b>NBD</b>) moiety to a selected fluorophore (Dye) through a sulfhydryl-labile ether linkage to afford nonfluorescent <b>NBD-O-Dye</b>. In the presence of GSH or Cys/Hcy, the ether bond is cleaved with the concomitant generation of both a nonfluorescent <b>NBD-S-R</b> derivative and a fluorescent dye having a characteristic intense emission band (<b>B1</b>). In the special case of Cys/Hcy, the <b>NBD-S-Cys/Hcy</b> cleavage product can undergo a further, rapid, intramolecular Smiles rearrangement to form a new, highly fluorescent <b>NBD-N-Cys/Hcy</b> compound (band <b>B2</b>); because of geometrical constraints, the GSH derived <b>NBD-S-GSH</b> derivative cannot undergo a Smiles rearrangement. Thus, the presence of a single <b>B1</b> or double <b>B1</b> + <b>B2</b> signature can be used to detect and differentiate GSH from Cys/Hcy, respectively. We demonstrate the broad applicability of our approach by including in our studies members of the Flavone, Bodipy, and Coumarin dye families. Particularly, single excitation wavelength could be applied for the probe <b>NBD-OF</b> in the detection of GSH over Cys/Hcy in both aqueous solution and living cells

Homoadamantane-Fused Tetrahydroquinoxaline as a Robust Electron-Donating Unit for High-Performance Asymmetric NIR Rhodamine Development

Rhodamines have emerged as a useful class of dye for bioimaging. However, intrinsic issues such as short emission wavelengths and small Stokes shifts limit their widespread applications in living systems. By taking advantage of the homoadamantane-fused tetrahydroquinoxaline (HFT) moiety as an electron donor, we developed a new class of asymmetric NIR rhodamine dyes, NNR1–7. These new dyes retained ideal photophysical properties from the classical rhodamine scaffold and showed large Stokes shifts (>80 nm) with improved chemo/photostability. We found that NNR1–7 specifically target cellular mitochondria with superior photobleaching resistance and improved tolerance for cell fixation compared to commercial mitochondria trackers. Based on NNR4, a novel NIR pH sensor (NNR4M) was also constructed and successfully applied for real-time monitoring of variations in lysosomal pH. We envision this design strategy would find broad applications in the development of highly stable NIR dyes with a large Stokes shift

Table Serum lipid Profiles of the rabbits in the four groups at week 16.

<p>Group A2 is Akt inhibitor triciribine treatment group, Group B2 is mTOR inhibitor rapamycin treatment group, Group C2 is mTOR-siRNA group and Group D2 is the control group.</p><p>TC, total cholesterol; TG, triglyceride; HDL, high density lipoprotein; LDL, low density lipoprotein.</p

Cytokines secreted by macrophages and effect of different inhibitors on Akt and mTOR molecules.

<p>(A, B) ELISA assay of cytokines in rabbit primary macrophage supernate showed an increased level of IL-10 and decreased level of IFN-γ in group A1 than E1. On the contrary, level of IL-10 was lower in group B1, C1 and D1 compared to group E1 whereas IFN-γ was higher. (C) QRT PCR showed that mRNA level declined in each group compared to group E1. (D, E) Western blot revealed that total Akt and total mTOR did not change among the five groups while phosphorylation of the Akt and mTOR reduced significantly in the experimental groups compared to the control. However, Akt in group C1 and D1 were hyperphosphorylated. A1: LY294002; B1: triciribine; C1: rapamycin; D1: mTOR-siRNA; E1: control. ^*<i>P</i><0.01 vs control, ^#<i>P</i><0.05 vs control.</p

Autophagy was induced in the presence of API-2, rapamycin and mTOR-siRNA respectively and inhibited in response to LY294002.

<p>Rabbit peritoneal macrophages were obtained to verify if blockage of PI3K, Akt, mTOR molecules respectively could promote autophagy. (A, B) Cell immunofluorescence staining of LC3-II among different groups (×400) showed that increased LC3-II punctate dots appeared in B1, C1 and D1 groups while decreased punctate dots in group A1 compared to group E1. (C) Expression of autophagy related gene Beclin 1 detected by QRT PCR showed remarkable higher Beclin 1 mRNA in group B1, C1 and D1 and lower in group A1 than group E1. (D) Western blot showed that expression of autophagy protein Beclin 1 and Atg5-Atg12 conjugation increased in group B1, C1 and D1 and decreased in group A1 than group E1. (E) Transmission electron microscope showed an intact nonpyknotic nucleus and numerous vacuoles, large cytoplasmic inclusions and myeline figure in the cytoplasm in group B1, C1 and D1, whereas group A1 and E1 did not display vacuolization but a little was observed (base level autophagy). Endoplasmic reticulum and mitochondria displayed varying degrees of swelling in group B1, C1 and D1. The “N” represents the nuclear. A1: LY294002; B1: triciribine; C1: rapamycin; D1: mTOR-siRNA; E1: control. ^*<i>P</i><0.01 vs control, ^#<i>P</i><0.05 vs control.</p

Effect of Pharmacological triggers in the experiment showed the rupture of plaque incidence higher in control group.

<p>IH staining and transmission electron microscope showed mTOR decreased and autophagy increased in experimental group. Pharmacological triggers were done by 0.15·kg⁻¹ of Chinese Russell's viper venom injecting intraperitoneally, 30 min later, 0.02 mg·kg⁻¹ histamine was injected intravenously. (A) Haematoxylin and eosin staining of the cross section of the abdominal aorta in a rabbit of group D2 showing plaque rupture and thrombosis. (B, C) IH staining showed mTOR decreased obviously in group A2, B2 and C2 than D2. (D) Transmission electron microscope analysis showed macrophage autophagy was enhanced in group A2, B2 and C2 compared to group D2. Arrows in picture B2, C2 and d2 represent vacuoles with cytoplasmic inclusions and myeline figure. The “N” represents the nuclear. (E) IH analysis of Atg5-Atg12 conjugation found percentage of positive-stain cells was significantly increased in group A2, B2 and C2 in comparison to group D2. A2: triciribine; B2: rapamycin; C2: mTOR-siRNA; D2: control; ^*<i>P</i><0.01 vs control, ^#<i>P</i><0.05 vs control.</p

Macrophage autophagy induced by drugs and mTOR-siRNA alleviated vulnerability index.

<p>The vulnerability index can be calculated as: (macrophage staining %+ lipid staining %)/(smooth muscle cell %+ collagen fiber %). So we conducted Sirius red (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090563#pone-0090563-g005" target="_blank">Fig. 5</a>) and oil red O staining together with IH for macrophages (RAM-11) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090563#pone-0090563-g005" target="_blank">Fig. 5</a>) and smooth muscle cells (α-SMA). (A, B) Oil Red O Staining was taken for lipid content evaluation and was expressed as a percentage verse the whole vessel lumen. Our result revealed much smaller plaque in aorta abdominals in group A2, B2 and C2 compared to group D2. (C) IH analysis of α-SMA amount did not detect difference in plaque area among the four groups. L represents for vascular lumen, P represents for plaque. (D) Vulnerability was calculated by macrophage, lipid, α-SMA and collagen. It was significantly lower in the experimental group than the control group. A2: triciribine; B2: rapamycin; C2: mTOR-siRNA; D2: control; ^*<i>P</i><0.01 vs control.</p

Macrophage autophagy promotes vulnerable plaque stability via decreased plaque burden and macrophages.

<p>IVUS measurement was taken to measure lumen area (LA), external elastic membrane area (EEMA), plaque area (PA) and plaque burden (PB) (PA =  EEMA – LA, PB% =  PA/EEMA ×100%). (A) IVUS image showed that the plaque area was huge in group D2 than that of A2, B2 and C2. (B) PA and PB calculated by LA and EEMA. We can see PA in the abdominal aorta in group A2, B2 and C2 was significantly lower than that in group D2, and PB% was remarkably reduced in group A2, B2 and C2 compared to group D2. However, the three groups did not differ in PB%. A2: triciribine; B2: rapamycin; C2: mTOR-siRNA; D2: control; ^*<i>P</i><0.01 vs control.</p

Sirius red staining for collagen and IH for macrophages in plaque.

<p>(A) The interstitial collagen content of aorta abdominals plaques was evaluated by Sirius red staining. Collagens are identified by birefringence under polarized light illumination following standard sirius red procedure. (B) IH for macrophage in plaque detected increased macrophages expressed in control group and less in group A2, B2 and C2. A2: triciribine; B2: rapamycin; C2: mTOR-siRNA; D2: control; ^*<i>P</i><0.01 vs control.</p