Semi-Quantitatively Designing Two-Photon High-Performance Fluorescent Probes for Glutathione S-Transferases.

Glutathione S-transferases (GSTs), detoxification enzymes that catalyze the addition of glutathione (GSH) to diverse electrophilic molecules, are often overexpressed in various tumor cells. While fluorescent probes for GSTs have often adopted the 2,4-dinitrobenzenesulfonyl (DNs) group as the receptor unit, they usually suffer from considerable background reaction noise with GSH due to excessive electron deficiency. However, weakening this reactivity is generally accompanied by loss of sensitivity for GSTs, and therefore, finely turning down the reactivity while maintaining certain sensitivity is critical for developing a practical probe. Here, we report a rational semiquantitative strategy for designing such a practical two-photon probe by introducing a parameter adopted from the conceptual density functional theory (CDFT), the local electrophilicity ω k , to characterize this reactivity. As expected, kinetic studies established ω k as efficient to predict the reactivity with GSH, and probe NI3 showing the best performance was successfully applied to detecting GST activities in live cells and tissue sections with high sensitivity and signal-to-noise ratio. Photoinduced electron transfer of naphthalimide-based probes, captured by femtosecond transient absorption for the first time and unraveled by theoretical calculations, also contributes to the negligible background noise.

A fluorophore's electron-deficiency does matter in designing high-performance near-infrared fluorescent probes.

The applications of most fluorescent probes available for Glutathione S-Transferases (GSTs), including NI3 which we developed recently based on 1,8-naphthalimide (NI), are limited by their short emission wavelengths due to insufficient penetration. To realize imaging at a deeper depth, near-infrared (NIR) fluorescent probes are required. Here we report for the first time the designing of NIR fluorescent probes for GSTs by employing the NIR fluorophore HCy which possesses a higher brightness, hydrophilicity and electron-deficiency relative to NI. Intriguingly, with the same receptor unit, the HCy-based probe is always more reactive towards glutathione than the NI-based one, regardless of the specific chemical structure of the receptor unit. This was proved to result from the higher electron-deficiency of HCy instead of its higher hydrophilicity based on a comprehensive analysis. Further, with caging of the autofluorescence being crucial and more difficult to achieve via photoinduced electron transfer (PET) for a NIR probe, the quenching mechanism of HCy-based probes was proved to be PET for the first time with femtosecond transient absorption and theoretical calculations. Thus, HCy2 and HCy9, which employ receptor units less reactive than the one adopted in NI3, turned out to be the most appropriate NIR probes with high-sensitivity and little nonenzymatic background noise. They were then successfully applied to detecting GST in cells, tissues and tumor xenografts in vivo. Additionally, unlike HCy2 with a broad isoenzyme selectivity, HCy9 is specific for GSTA1-1, which is attributed to its lower reactivity and the higher effectiveness of GSTA1-1 in stabilizing the active intermediate via H-bonds based on docking simulations.

Correction: A versatile two-photon fluorescent probe for ratiometric imaging E. coliß-galactosidase in live cells and in vivo.

Correction for 'A versatile two-photon fluorescent probe for ratiometric imaging E. coliß-galactosidase in live cells and in vivo' by Xue-Xiang Zhang et al., Chem. Commun., 2016, 52, 8283-8286.

A versatile two-photon fluorescent probe for ratiometric imaging E. coliß-galactosidase in live cells and in vivo.

We have described the design, synthesis, spectroscopy and biological applications of NI-ßGal, a versatile fluorescent probe to detect E. coliß-galactosidase in live cells and mice sensitively and directly, which holds great promise for its application in biomedical research such as gene therapy for cancer in the future.

[Clinical impacts of highly active antiretroviral therapy on patients of hemophilia combined with acquired immunodeficiency syndrome: 6-year follow-up of 39 cases].

OBJECTIVE: To evaluate the impact of highly active antiretroviral therapy (HAART) on the haemorrhage status, joint function, and physical ability of the patients of hemophilia combined with acquired immunodeficiency syndrome (AIDS). METHODS: Thirty-nine hemophilia A/AIDS patients, all male, aged (40+/-13), underwent HAART and followed up for 6 years from 2002 to 2008 to observe the yearly hospital visit time, bleeding time, transfusion times, amount of factor VIII transfusion, VIII: C level, physical ability, and joint function. Flow cytometry was used to count the CD4+ T cells, and bDNA assay was used to examine the HIV virus load. RESULTS: The average hospital visit time, bleeding time, transfusion time, and amount of factor VIII transfusion, hemoglobin, white blood cell count, and platelet count before HAART were not significantly different from those after treatment (all P>0.05); only one case showed moderate decrease in VIII: C level, and another one case showed slight decline in physical ability and joint function. The serum HIV RNA load decreased from (4.8+/-1.0) log copies/ml before HAART to (2.4+/-1.0) log copies/ml (P<0.05) and the CD4+ T cell count raised from 183+/-97/mm3 to 456+/-157/mm3 (P<0.05) after HAART. CONCLUSION: HAART has no obvious impact on the haemorrhage status, joint function, and physical ability in hemophilia A/AIDS patients, however, it is effective to inhibit HIV replication and raise CD4+ T cell number which indicates that HAART therapy is positive for immune recovery.

[Peg-interferon alfa-2a and highly active antiretroviral drug therapy on hepatitis C patients with AIDS].

OBJECTIVE: To evaluate the clinical effect and side-effect of peg-interferon alfa-2a (PEG-IFN alfa-2a) and highly active antiretroviral therapy (HAART) for patients infected with hepatitis C virus (HCV) and co-infected with human immunodeficiency virus (HIV). METHODS: Twenty-two patients with HCV/HIV co-infection received highly active antiretroviral therapy initially; after their CD4 lymphocyte counts rose to over 0.20x10(9)/L, they were separated into two groups: one group with CD4 lymphocytes over 0.35x10(9)/L (high group) and one group with CD4 lymphocytes below 0.35x10(9)/L (low group). Both groups were given 180 microg of PEG-IFN alfa-2a every week intramuscularly. HCV RNA and HIV RNA loads, blood cell and CD4 lymphocyte counts, and liver functions were routinely examined. RESULTS: After 12, 24 and 48 weeks of PEG-IFN alfa-2a therapy, mean HCV RNA loads reduced 2.0650 log10 copies/ml (t=3.8733), 2.9146 log10 copies/ml (t=7.6741) and 2.4315 log10 copies/ml (t=5.8202) from the baseline at week 0 in the 13 patients in the high group, and reduced 1.1522 log10 copies/ml (t = 2.8937), 1.4189 log10 copies/ml (t=2.4422) and 1.1167 log10 (t=1.1261) in the 8 patients of the low group. However, there was no significant difference between the early viral response rate (EVR) and the end of treatment viral response rate (ETVR) of the two groups. In the high group, the white blood cell count was lower at 24 weeks than the base line (t=2.4700), and the blood platelet count was lower both at 24 and 48 weeks than the base line (t=2.3273 and t=3.6149). CONCLUSIONS: PEG-IFN alfa-2a can effectively reduce HCV RNA loads in patients with HCV-/HIV co-infection, and the inhibition rate in patients with higher CD4 lymphocyte counts is better. The EVR and ETVR of the two groups of patients show similar results after the treatment. PEG-IFN alfa-2a can reduce the white blood cell counts and the blood platelet counts in the peripheral blood.

Effects of (-)stepholidine in animal models for schizophrenia.

AIM: (-)Stepholidine (SPD) is an active ingredient of the Chinese herb Stephania intermedia, which binds to the dopamine D(1) and D(2) like receptors. Biochemical, electrophysiological and behavioural experiments have provided strong evidence that SPD is both a D(1) and a D(2) antagonist, which could make SPD a unique antipsychotic drug. The present study aimed to investigate the antipsychotic properties of SPD in two animal models for schizophrenia. METHODS: The effects of SPD, clozapine and haloperidol in increasing forelimb and hindlimb retraction time in the paw test and in reversing the apomorphine and MK801-induced disruption of prepulse inhibition was investigated. RESULTS: In the paw test, clozapine and SPD increased the hindlimb retraction time, with only a marginal effect on the forelimb retraction time, whereas haloperidol potently increased both. In the prepulse inhibition paradigm, all three drugs reverse the apomorphine-induced disruption in prepulse inhibition, while none of the drugs could reverse the MK801-induced disruption. SPD even slightly, but significantly, potentiated the effects of MK801. CONCLUSION: The data show that SPD showed antipsychotic-like effects in both the prepulse inhibition paradigm and in the paw test. Moreover, the results of the paw test suggest that SPD has an atypical character with a relatively small potency to induce extrapyramidal side effects.

Dendritic glutamate-induced bursting in the prefrontal cortex: further characterization and effects of phencyclidine.

To understand the role of N-methyl-d-aspartate (NMDA) receptors in the prefrontal cortex (PFC) and to investigate how the psychotomimetic drug phencyclidine (PCP) may alter PFC function, we made whole-cell recordings from PFC neurons in rat brain slices. Our result showed that most deep layer pyramidal neurons in the PFC were regular spiking cells. They could fire repetitive bursts, however, when activated by glutamate focally applied to the apical dendrite. Application of NMDA to the same dendritic spot also induced bursting, whereas application of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) evoked single spikes only. Coapplication of AMPA with NMDA evoked more single spikes and decreased NMDA-induced bursting. Experiments with NMDA and AMPA antagonists further showed that dendritic glutamate (dGlu)-induced bursting required NMDA receptor activation and was enhanced when AMPA receptors were blocked. At subanesthetic concentrations, PCP decreased dGlu-induced bursting and altered the temporal characteristics of the bursts by decreasing spikes per burst and increasing interspike intervals within bursts. The latter two changes were not observed when AMPA receptors were blocked, suggesting that they are secondary to the increased AMPA receptor contribution to glutamate responses evoked in the presence of PCP. These results suggest that NMDA receptors are essential for PFC pyramidal cells to fire in bursts in response to dGlu input and that PCP suppresses dGlu-induced bursting. Since bursting is necessary for pyramidal cells to activate GABA interneurons, the suppression effect of PCP may further lead to a weakening of the connections from pyramidal cells and GABA interneurons, thereby contributing to PCP's psychotomimetic effects.