Allosteric Modulatory Effects of SRI-20041 and SRI-30827 on Cocaine and HIV-1 Tat Protein Binding to Human Dopamine Transporter

Dopamine transporter (DAT) is the target of cocaine and HIV-1 transactivator of transcription (Tat) protein. Identifying allosteric modulatory molecules with potential attenuation of cocaine and Tat binding to DAT are of great scientific and clinical interest. We demonstrated that tyrosine 470 and 88 act as functional recognition residues in human DAT (hDAT) for Tat-induced inhibition of DA transport and transporter conformational transitions. Here we investigated the allosteric modulatory effects of two allosteric ligands, SRI-20041 and SRI-30827 on cocaine binding on wild type (WT) hDAT, Y470 H and Y88 F mutants. Effect of SRI-30827 on Tat-induced inhibition of [3H]WIN35,428 binding was also determined. Compared to a competitive DAT inhibitor indatraline, both SRI-compounds displayed a similar decrease (30%) in IC50 for inhibition of [3H]DA uptake by cocaine in WT hDAT. The addition of SRI-20041 or SRI-30827 following cocaine slowed the dissociation rate of [3H]WIN35,428 binding in WT hDAT relative to cocaine alone. Moreover, Y470H and Y88F hDAT potentiate the inhibitory effect of cocaine on DA uptake and attenuate the effects of SRI-compounds on cocaine-mediated dissociation rate. SRI-30827 attenuated Tat-induced inhibition of [3H]WIN35,428 binding. These observations demonstrate that tyrosine 470 and 88 are critical for allosteric modulatory effects of SRI-compounds on the interaction of cocaine with hDAT

Role of Histidine 547 of Human Dopamine Transporter in Molecular Interaction with HIV-1 Tat and Dopamine Uptake

HIV-1 Tat plays an important role in HIV-associated neurocognitive disorders (HAND) by disrupting neurotransmission including dopamine uptake by human dopamine transporter (hDAT). Previous studies have demonstrated that HIV-1 Tat directly binds to hDAT and some amino-acid mutations that attenuate the hDAT-Tat binding also significantly decreased dopamine uptake activity of hDAT. This combined computational-experimental study demonstrates that histidine-547 (H547) of hDAT plays a crucial role in the hDAT-Tat binding and dopamine uptake by hDAT, and that the H547A mutation can not only considerably attenuate Tat-induced inhibition of dopamine uptake, but also significantly increase the Vmax of hDAT for dopamine uptake. The finding of such an unusual hDAT mutant capable of both increasing the Vmax of hDAT for dopamine uptake and disrupting the hDAT-Tat binding may provide an exciting knowledge basis for development of novel concepts for therapeutic treatment of the HAND

Molecular Mechanism: The Human Dopamine Transporter Histidine 547 Regulates Basal and HIV-1 Tat Protein-Inhibited Dopamine Transport

Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-1-associated neurocognitive disorders. HIV-1 Tat protein increases synaptic dopamine (DA) levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Through integrated computational modeling prediction and experimental validation, we identified that histidine547 on human DAT (hDAT) is critical for regulation of basal DA uptake and Tat-induced inhibition of DA transport. Compared to wild type hDAT (WT hDAT), mutation of histidine547 (H547A) displayed a 196% increase in DA uptake. Other substitutions of histidine547 showed that DA uptake was not altered in H547R but decreased by 99% in H547P and 60% in H547D, respectively. These mutants did not alter DAT surface expression or surface DAT binding sites. H547 mutants attenuated Tat-induced inhibition of DA transport observed in WT hDAT. H547A displays a differential sensitivity to PMA- or BIM-induced activation or inhibition of DAT function relative to WT hDAT, indicating a change in basal PKC activity in H547A. These findings demonstrate that histidine547 on hDAT plays a crucial role in stabilizing basal DA transport and Tat-DAT interaction. This study provides mechanistic insights into identifying targets on DAT for Tat binding and improving DAT-mediated dysfunction of DA transmission

Mutational Effects of Human Dopamine Transporter at Tyrosine88, Lysine92, and Histidine547 on Basal and HIV-1 Tat-Inhibited Dopamine Transport

Dysregulation of dopaminergic system induced by HIV-1 Tat protein-mediated direct inhibition of the dopamine transporter (DAT) has been implicated as a mediating factor of HIV-1 associated neurocognitive disorders. We have reported that single point mutations on human DAT (hDAT) at tyrosine88 (Y88F), lysine92 (K92M), and histidine547 (H547A) differentially regulate basal dopamine uptake but diminish Tat-induced inhibition of dopamine uptake by changing dopamine transport process. This study evaluated the effects of double (Y88F/H547A) and triple (Y88F/K92M/H547A) mutations on basal dopamine uptake, Tat-induced inhibition of DAT function, and dynamic transport process. Compared to wild-type hDAT, the Vmax values of [3H]Dopamine uptake were increased by 96% in Y88F/H547A but decreased by 97% in Y88F/K92M/H547A. [3H]WIN35,428 binding sites were not altered in Y88F/H547A but decreased in Y88F/K92M/H547A. Y88F/H547A mutant attenuated Tat-induced inhibition of dopamine uptake observed in wild-type hDAT. Y88F/H547A displayed an attenuation of zinc-augmented [3H]WIN35,428 binding, increased basal dopamine efflux, and reduced amphetamine-induced dopamine efflux, indicating this mutant alters transporter conformational transitions. These findings further demonstrate that both tyrosine88 and histidine547 on hDAT play a key role in stabilizing basal dopamine transport and Tat-DAT integration. This study provides mechanistic insights into developing small molecules to block multiple sites in DAT for Tat binding

Adjuvant Chemotherapy Versus Adjuvant Concurrent Chemoradiotherapy After Radical Surgery for Early-Stage Cervical Cancer: A Randomized, Non-Inferiority, Multicenter Trial

We conducted a prospective study to assess the non-inferiority of adjuvant chemotherapy alone versus adjuvant concurrent chemoradiotherapy (CCRT) as an alternative strategy for patients with early-stage (FIGO 2009 stage IB-IIA) cervical cancer having risk factors after surgery. The condition was assessed in terms of prognosis, adverse effects, and quality of life. This randomized trial involved nine centers across China. Eligible patients were randomized to receive adjuvant chemotherapy or CCRT after surgery. The primary end-point was progression-free survival (PFS). From December 2012 to December 2014, 337 patients were subjected to randomization. Final analysis included 329 patients, including 165 in the adjuvant chemotherapy group and 164 in the adjuvant CCRT group. The median follow-up was 72.1 months. The three-year PFS rates were both 91.9%, and the five-year OS was 90.6% versus 90.0% in adjuvant chemotherapy and CCRT groups, respectively. No significant differences were observed in the PFS or OS between groups. The adjusted HR for PFS was 0.854 (95% confidence interval 0.415-1.757; P = 0.667) favoring adjuvant chemotherapy, excluding the predefined non-inferiority boundary of 1.9. The chemotherapy group showed a tendency toward good quality of life. In comparison with post-operative adjuvant CCRT, adjuvant chemotherapy treatment showed non-inferior efficacy in patients with early-stage cervical cancer having pathological risk factors. Adjuvant chemotherapy alone is a favorable alternative post-operative treatment

Investigation of Helium Behavior in RAFM Steel by Positron Annihilation Doppler Broadening and Thermal Desorption Spectroscopy

The behavior of helium in reduced-activation ferritic/martensitic steels was investigated systematically with positron annihilation Doppler broadening measurement and thermal desorption spectroscopy. Specimens were irradiated with helium ions with different energies to various fluences at different temperatures. A threshold fluence was observed above which the rate of formation and growth of helium bubbles dramatically increased. Irradiation at higher temperature could suppress the formation and growth of HenVm clusters with low binding energies and enhance that of helium bubbles and HenVm clusters with high binding energies. Different changes of S parameters were observed in various depth after the irradiation temperature was increased from 523 K to 723 K. Irradiation of 18 keV-He+ enhanced the growth of HenVm clusters and helium bubbles compared with 100 keV-He+ irradiation. A possible mechanism is discussed

Irradiation Hardening and Microstructure Study of MAX-Phase-Dispersion-Strengthened Vanadium Alloy under Self-Ion Irradiation

V-4Cr-4Ti alloy is one of the candidate structural materials for future fusion reactors due to its desirable characteristics. In our previous research, MAX-phase-dispersion-strengthened vanadium alloy (V-4Cr-4Ti-1.5Y-0.3Ti3SiC2), prepared through mechanical alloying, showed excellent thermal stability and creep resistance and was expected to have good radiation resistance. This study investigates the effects of 2.5 MeV V2+ ion irradiation on V-4Cr-4Ti-1.5Y-0.3Ti3SiC2 and V-4Cr-4Ti alloys at 500 °C, with peak damage of 0.8, 3.5, and 6.1 dpa. Transmission electron microscopy and nanoindentation were used to examine the changes in microstructure and hardness before and after irradiation. The microscopic analysis reveals that dispersed nanoparticles maintained good stability under irradiation. Defect clusters grow with increasing irradiation doses in both materials. The nanoindentation results show that V-4Cr-4Ti-1.5Y-0.3Ti3SiC2 has higher initial hardness and lower irradiation hardening, indicating better resistance to radiation hardening than V-4Cr-4Ti. This research serves as a valuable reference for the assessment of the irradiation resistance of Ti3SiC2-dispersion-strengthened V-4Cr-4Ti alloy

Development of a Radio-Frequency Quadrupole Accelerator for the HL-2A/2M Tokamak Diagnostic System

In order to figure out the migration and deposition of impurities on the first wall of HL-2A/2M tokamak, Peking University and Southwestern Institute of Physics are co-developing a deuteron RFQ as part of the in situ ion-beam diagnostic for the material. The RFQ, which operates at 162.5 MHz, is designed to accelerate a 10-mA deuteron beam from 40 keV up to 1.5 MeV. Key design considerations and the final design parameters are presented. The RFQ has been conditioned at a 1% duty factor for 80 h at RF cavity power of 55 kW. The specific shunt impedance of the cavity is 221 kΩ·m by measuring the bremsstrahlung spectrum. The intrinsic Q-value after the high-power tests measured by the Ring-Down method is 13,780. Beam commissioning has been taken place during the first half of 2021, and the beam measurements include beam current and energy of 2H+ ion. A 10 mA 2H+ beam was successfully accelerated through the RFQ

Evolution of Dislocation Loops Induced by Different Hydrogen Irradiation Conditions in Reduced-Activation Martensitic Steel

Hydrogen can be induced in various ways into reduced-activation ferritic/martensitic (RAFM) steels when they are used as structural materials for advanced nuclear systems. However, because of the fast diffusion of hydrogen in metals, the effect of hydrogen on the evolution of irradiation-induced defects was almost neglected. In the present work, the effect of hydrogen on the evolution of dislocation loops was investigated using a transmission electron microscope. Specimens of reduced-activation ferritic/martensitic (RAFM) steels were irradiated with hydrogen ions to 5 × 1020 H+ • m−2 at 523⁻823 K, and to 1 × 1020 H+ • m−2 − 5 × 1020 H+ • m−2 at 723 K. The experimental results reveal that there is an optimum temperature for dislocation loop growth, which is ~723 K, and it is greater than the reported values for neutron irradiations. Surprisingly, the sizes of the loops produced by hydrogen ions, namely, 93 nm and 286 nm for the mean and maximum value, respectively, at the peak dose of 0.16 dpa under 723 K, are much larger than that produced by neutrons and heavy ions at the same damage level and temperature. The results indicate that hydrogen could enhance the growth of loops. Moreover, 47.3% 1 2   a0 <111> and 52.7% a0 <100> loops were observed at 523 K, but 1 2   a0 <111> loops disappeared and only a0 <100> loops existed above 623 K. Compared with the neutron and ion irradiations, the presence of hydrogen promoted the formation of a0 <100> loops

Mutations of tyrosine 467 in the human norepinephrine transporter attenuate HIV-1 Tat-induced inhibition of dopamine transport while retaining physiological function.

Dysregulation of dopaminergic transmission induced by the HIV-1 transactivator of transcription (Tat) has been implicated as a central factor in the development of HIV-1 associated neurocognitive disorders (HAND). We have demonstrated that the tyrosine470 residue of the human dopamine transporter (hDAT) plays a critical role in Tat-hDAT interaction. Based on the computational modeling predictions, the present study sought to examine the mutational effects of the tyrosine467 residue of the human norepinephrine transporter (hNET), a corresponding residue of the hDAT tyrosine470, on Tat-induced inhibition of reuptake of dopamine through the hNET. Mutations of the hNET tyrosine467 to a histidine (Y467H) or a phenylalanine (Y467F) displayed similar kinetic properties of reuptake of [3H]dopamine and [3H]norepinephrine in PC12 cells expressing wild-type hNET and its mutants. Compared to wild-type hNET, neither of Y467H or Y467F altered Bmax and Kd values of [3H]WIN35,428 binding, whereas Y467H but not Y467F decreased the Bmax of [3H]nisoxetine binding without changes in Kd. Y467H also increased the affinity of nisoxetine for inhibiting [3H]dopamine uptake relative to wild-type hNET. Recombinant Tat1-86 (140 nM) induced a significant reduction of [3H]dopamine uptake in wild-type hNET, which was attenuated in both Y467H and Y467F. Compared to wild-type hNET, neither Y467H or Y467F altered [3H]dopamine efflux in CHO cells expressing WT hNET and mutants, whereas Y467F but not Y467H decreased [3H]MPP+ efflux. These results demonstrate tyrosine467 as a functional recognition residue in the hNET for Tat-induced inhibition of dopamine transport and provide a novel insight into the molecular basis for developing selective compounds that target Tat-NET interactions in the context of HAND