Inhibition of activity of GABA transporter GAT1 by δ-opioid receptor

Analgesia is a well-documented effect of acupuncture. A critical role in pain sensation plays the nervous system, including the GABAergic system and opioid receptor (OR) activation. Here we investigated regulation of GABA transporter GAT1 by δOR in rats and in Xenopus oocytes. Synaptosomes of brain from rats chronically exposed to opiates exhibited reduced GABA uptake, indicating that GABA transport might be regulated by opioid receptors. For further investigation we have expressed GAT1 of mouse brain together with mouse δOR and μOR in Xenopus oocytes. The function of GAT1 was analyzed in terms of Na(+)-dependent [(3)H]GABA uptake as well as GAT1-mediated currents. Coexpression of δOR led to reduced number of fully functional GAT1 transporters, reduced substrate translocation, and GAT1-mediated current. Activation of δOR further reduced the rate of GABA uptake as well as GAT1-mediated current. Coexpression of μOR, as well as μOR activation, affected neither the number of transporters, nor rate of GABA uptake, nor GAT1-mediated current. Inhibition of GAT1-mediated current by activation of δOR was confirmed in whole-cell patch-clamp experiments on rat brain slices of periaqueductal gray. We conclude that inhibition of GAT1 function will strengthen the inhibitory action of the GABAergic system and hence may contribute to acupuncture-induced analgesia

LMBAO: A Landmark Map for Bundle Adjustment Odometry in LiDAR SLAM

LiDAR odometry is one of the essential parts of LiDAR simultaneous localization and mapping (SLAM). However, existing LiDAR odometry tends to match a new scan simply iteratively with previous fixed-pose scans, gradually accumulating errors. Furthermore, as an effective joint optimization mechanism, bundle adjustment (BA) cannot be directly introduced into real-time odometry due to the intensive computation of large-scale global landmarks. Therefore, this letter designs a new strategy named a landmark map for bundle adjustment odometry (LMBAO) in LiDAR SLAM to solve these problems. First, BA-based odometry is further developed with an active landmark maintenance strategy for a more accurate local registration and avoiding cumulative errors. Specifically, this paper keeps entire stable landmarks on the map instead of just their feature points in the sliding window and deletes the landmarks according to their active grade. Next, the sliding window length is reduced, and marginalization is performed to retain the scans outside the window but corresponding to active landmarks on the map, greatly simplifying the computation and improving the real-time properties. In addition, experiments on three challenging datasets show that our algorithm achieves real-time performance in outdoor driving and outperforms state-of-the-art LiDAR SLAM algorithms, including Lego-LOAM and VLOM.Comment: 9 pages, 3 tables, 6 figure

Ligand Selectivity in the Recognition of Protoberberine Alkaloids by Hybrid-2 Human Telomeric G-Quadruplex: Binding Free Energy Calculation, Fluorescence Binding, and NMR Experiments

The human telomeric G-quadruplex (G4) is an attractive target for developing anticancer drugs. Natural products protoberberine alkaloids are known to bind human telomeric G4 and inhibit telomerase. Among several structurally similar protoberberine alkaloids, epiberberine (EPI) shows the greatest specificity in recognizing the human telomeric G4 over duplex DNA and other G4s. Recently, NMR study revealed that EPI recognizes specifically the hybrid-2 form human telomeric G4 by inducing large rearrangements in the 50-flanking segment and loop regions to form a highly extensive four-layered binding pocket. Using the NMR structure of the EPI-human telomeric G4 complex, here we perform molecular dynamics free energy calculations to elucidate the ligand selectivity in the recognition of protoberberines by the human telomeric G4. The MM-PB(GB)SA (molecular mechanics-Poisson Boltzmann/Generalized Born) Surface Area) binding free energies calculated using the Amber force fields bsc0 and OL15 correlate well with the NMR titration and binding affinity measurements, with both calculations correctly identifying the EPI as the strongest binder to the hybrid-2 telomeric G4 wtTel26. The results demonstrated that accounting for the conformational flexibility of the DNA-ligand complexes is crucially important for explaining the ligand selectivity of the human telomeric G4. While the MD-simulated (molecular dynamics) structures of the G-quadruplex-alkaloid complexes help rationalize why the EPI-G4 interactions are optimal compared with the other protoberberines, structural deviations from the NMR structure near the binding site are observed in the MD simulations. We have also performed binding free energy calculation using the more rigorous double decoupling method (DDM); however, the results correlate less well with the experimental trend, likely due to the difficulty of adequately sampling the very large conformational reorganization in the G4 induced by the protoberberine binding

In-line microfiber-assisted Mach-Zehnder interferometer for microfluidic highly sensitive measurement of salinity

We present a microfluidic U-shaped micro-cavity sensor by splicing a segment of microfiber of a few hundred micrometers in length tapered from a single-mode fiber (SMF) between two SMFs with predesigned lateral offset for highly sensitive salinity measurement. The proposed sensing probe serves as an in-line microfiber-assisted Mach-Zehnder interferometer (MAMZI) with an ultra-high refractive index sensitivity of 104 nm/RIU. Three Mach-Zehnder interferometer structures with different cavity lengths of 351.82 ?m, 242.56 ?m and 181.31 ?m are fabricated, by which microfluidic sensing systems are established for in-line measurement of sodium chloride (NaCl) solution. Experimental results indicate that the detection limit of NaCl solution is as low as 4×10-3 wt% and the response time is less than 15 s, which would make the MAMZI-based microfluidic measuring system play an important role in label-free biological and chemical detection applications

The mechanism of H171T resistance reveals the importance of Nδ-protonated His171 for the binding of allosteric inhibitor BI-D to HIV-1 integrase

Background: Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an important new class of anti-HIV-1 agents. ALLINIs bind at the IN catalytic core domain (CCD) dimer interface occupying the principal binding pocket of its cellular cofactor LEDGF/p75. Consequently, ALLINIs inhibit HIV-1 IN interaction with LEDGF/p75 as well as promote aberrant IN multimerization. Selection of viral strains emerging under the inhibitor pressure has revealed mutations at the IN dimer interface near the inhibitor binding site. Results: We have investigated the effects of one of the most prevalent substitutions, H171T IN, selected under increasing pressure of ALLINI BI-D. Virus containing the H171T IN substitution exhibited an ~68-fold resistance to BI-D treatment in infected cells. These results correlated with ~84-fold reduced affinity for BI-D binding to recombinant H171T IN CCD protein compared to its wild type (WT) counterpart. However, the H171T IN substitution only modestly affected IN-LEDGF/p75 binding and allowed HIV-1 containing this substitution to replicate at near WT levels. The x-ray crystal structures of BI-D binding to WT and H171T IN CCD dimers coupled with binding free energy calculations revealed the importance of the Nδ- protonated imidazole group of His171 for hydrogen bonding to the BI-D tert-butoxy ether oxygen and establishing electrostatic interactions with the inhibitor carboxylic acid, whereas these interactions were compromised upon substitution to Thr171. Conclusions: Our findings reveal a distinct mechanism of resistance for the H171T IN mutation to ALLINI BI-D and indicate a previously undescribed role of the His171 side chain for binding the inhibitor. Electronic supplementary material The online version of this article (doi:10.1186/s12977-014-0100-1) contains supplementary material, which is available to authorized users

A New Class of Allosteric HIV-1 Integrase Inhibitors Identified by Crstallographic Fragment Screening of the Catalytic Core Domain

HIV-1 integrase (IN) is essential for virus replication and represents an important multifunctional therapeutic target. Recently discovered quinoline-based allosteric IN inhibitors (ALLINIs) potently impair HIV-1 replication and are currently in clinical trials. ALLINIs exhibit a multimodal mechanism of action by inducing aberrant IN multimerization during virion morphogenesis and by competing with IN for binding to its cognate cellular cofactor LEDGF/p75 during early steps of HIV-1 infection. However, quinoline-based ALLINIs impose a low genetic barrier for the evolution of resistant phenotypes, which highlights a need for discovery of second-generation inhibitors. Using crystallographic screening of a library of 971 fragments against the HIV-1 IN catalytic core domain (CCD) followed by a fragment expansion approach, we have identified thiophenecarboxylic acid derivatives that bind at the CCD-CCD dimer interface at the principal lens epithelium-derived growth factor (LEDGF)/p75 binding pocket. The most active derivative (5) inhibited LEDGF/p75-dependent HIV-1 IN activity in vitro with an IC50 of 72 μm and impaired HIV-1 infection of T cells at an EC50 of 36 μm. The identified lead compound, with a relatively small molecular weight (221 Da), provides an optimal building block for developing a new class of inhibitors. Furthermore, although structurally distinct thiophenecarboxylic acid derivatives target a similar pocket at the IN dimer interface as the quinoline-based ALLINIs, the lead compound, 5, inhibited IN mutants that confer resistance to quinoline-based compounds. Collectively, our findings provide a plausible path for structure-based development of second-generation ALLINIs

Dynamic Surveillance of Mosquitoes and Their Viromes in Wuhan During 2020

Mosquitoes are medically important arthropod vectors that harbor a variety of viruses. Geography and climate are known to be associated with variations in mosquito density, species and viromes. Our study investigated the dynamic changes in mosquito populations, species compositions and viromes in a regularly disinfected environment in Wuhan, China, during 2020. Traps were set in different mosquito habitats, including an urban residential area, two hospitals, a scenic area and a pig farm in a rural region between April and October of 2020. The collected mosquitoes were subjected to morphological identification, RT-qPCR and metagenomic sequencing. A total of 2345 adult mosquitoes were collected. Culex mosquitoes were dominant in both urban regions (90.32%, 1538/1703) and the pig farm (54.98%, 353/642). In RT-qPCR screening, the prevalence of Banna virus was 15% and 3% in mosquitoes from the urban area and the pig farm, respectively, whereas no Japanese encephalitis virus was detected. Culex viromes showed dynamic changes during the collection period. Several mosquito-specific viruses, such as Culex flavivirus, Alphamesonivirus 1, Hubei mosquito virus 2 and Hubei mosquito virus 4, showed seasonal changes and unimodal increases or declines. Other mosquito-specific viruses, such as Wuhan mosquito virus 6, Hubei virga-like virus 2 and Zhejiang mosquito virus 3, were stable in all collected Culex and are potential members of the core viromes. This study improves understanding of the dynamic composition of mosquito species and the viromes that they carry, and provides useful information for guiding mosquito control and mosquito-borne disease prevention strategies

Distinguishing Binders from False Positives by Free Energy Calculations: Fragment Screening Against the Flap Site of HIV Protease

Molecular docking is a powerful tool used in drug discovery and structural biology for predicting the structures of ligand–receptor complexes. However, the accuracy of docking calculations can be limited by factors such as the neglect of protein reorganization in the scoring function; as a result, ligand screening can produce a high rate of false positive hits. Although absolute binding free energy methods still have difficulty in accurately rank-ordering binders, we believe that they can be fruitfully employed to distinguish binders from nonbinders and reduce the false positive rate. Here we study a set of ligands that dock favorably to a newly discovered, potentially allosteric site on the flap of HIV-1 protease. Fragment binding to this site stabilizes a closed form of protease, which could be exploited for the design of allosteric inhibitors. Twenty-three top-ranked protein–ligand complexes from AutoDock were subject to the free energy screening using two methods, the recently developed binding energy analysis method (BEDAM) and the standard double decoupling method (DDM). Free energy calculations correctly identified most of the false positives (≥83%) and recovered all the confirmed binders. The results show a gap averaging ≥3.7 kcal/mol, separating the binders and the false positives. We present a formula that decomposes the binding free energy into contributions from the receptor conformational macrostates, which provides insights into the roles of different binding modes. Our binding free energy component analysis further suggests that improving the treatment for the desolvation penalty associated with the unfulfilled polar groups could reduce the rate of false positive hits in docking. The current study demonstrates that the combination of docking with free energy methods can be very useful for more accurate ligand screening against valuable drug targets

Geopolymerization of illite/smectite (Friedland Clay) - An investigation of reaction processes, microstructure and strength development

Using geopolymers can reduce significant amounts of CO2-emissions during the production compared to Portland cement. Although illite/smectite clays are very abundant on earths crust and rich in SiO2 and Al2O3, studies of their geopolymerization potential are rare. Thus, the illite/smectite clay of Friedland (NE Germany) was calcined (850 °C) and ground to form a reactive metaclay and then mixed with synthetic gibbsite (to test the effect of Al-concentration) and 6 molar NaOH or KOH, in order to study their geopolymerization at 25, 50 and 75 °C within 28 days. The raw clay, the precursors, and the geopolymers were characterized by XRF, XRD, SEM-EDX, Flame-AAS, nitrogen adsorption and compressive strength test. 25 °C was too low to initiate the geopolymerization of illite/smectite. Increasing the curing temperature increased the reactivity of meta-illite/smecite. Si and Al dissolution was confined to the first 24 h, followed by the hardening of the geopolymers within 28 days. At 50°C, KOH-activation formed amorphous and mesoporous aluminosilicates, which significantly cemented the particles and agglomerates of the metaclay. Consequently, geopolymers with high compression strength (~38 N/mm2) were formed. Adding 10 wt% Gibbsite (precursor Si/Al = 2.1) to the metaclay strengthened the formation of amorphous aluminosilicates and increased the compression strength of the geopolymer by 20 % from 38 - 45 N/mm2. At 75 °C, the reactivity of the metaclay in NaOH was higher than in KOH. NaOHactivation at that temperature formed geopolymers with high compression strength (~30 N/mm2) due to the cementation by microporous phillipsite (K-, Na-zeolite) crystals. Thus, alkali-activation of the calcined and ground meta-illite/smectite from Friedland form high strength geopolymers under hydrothermal conditions.Im Vergleich zu Portlandzement kann bei der Herstellung von Geopolymeren erhebliche Mengen an CO2-Emission eingespart werden. Illit/Smektit ist ein sehr häufig vorkommendes Tonmaterial auf der Erdoberfläche und aufgrund des hohen Gehalts an SiO2 und Al2O3 sehr gut für die Geopolymersynthese geeignet. Trotzdem gibt es nur sehr wenige Untersuchungen zur Geopolymerisierung von Illit/Smektit. Deshalb wurde in dieser Arbeit die Geopolymerisierung des Illit/Smektit-Tons aus Friedland (Mecklenburg-Vorpommern Deutschland) untersucht. Dabei wurde der Illit/Smektit- Ton bei 850 °C erhitzt und anschließend aufgemahlen, um einen reaktiven Metaton (Meta-Illit/Smektit) herzustellen. Zusätzlich wurde Gibbsit zum Metaton gegeben, um den Einfluß der Al-Konzentration des Ausgangsmaterials auf die Geopolymerisierung von Meta-Illit/Smektit bei 25, 50 und 75 °C innerhalb von 28 Tagen zu untersuchen. 6 molare NaOH bzw. KOH wurde für die Alkaliaktivierung verwendet. Das Ausgangsmaterial und die Geopolymere wurden mit XRF, XRD, SEM-EDX, Flammen-AAS, N2-Adsorption und Druckfestigkeitsprüfung analysiert. Es zeigte sich, daß 25 °C zu niedrig ist, um die Geopolymerisierung von Meta-Illit/Smektit zu initiieren. Die Alkaliaktivierung beschränkt sich auf die ersten 24 h, wonach die Aushärtung des Geopolymers innerhalb von 28 Tagen erfolgt. Bei 50 °C wurde die KOH-Aktivierung begünstigt, bei der amorphe und mesoporöse Aluminosilikate sich bildeten und den Zementköper fest zementierte. Die Zugabe von 10 Gew.% Gibbsit konnte das Bilden der amorphen Aluminosilikate verstärken und die Druckfestigkeit des Geopolymers um ~20 % von 38 - 45 N/mm2 erhöhen. Bei 75 °C zeigte der Meta-Illit/Smektit in NaOH eine höhere Reaktivität als in KOH. Bei dieser Temperatur bildete die NaOH-Aktivierung mikroporöse Phillipsit Kristalle (K-, Na-Zeolith), die den Zementkörper fest zementierte und Geopolymere mit einer Druckfesitigkeit von ~30 N/mm2 bildeten. Gibbsit zeigte keinen signifikanten Einfluß auf die NaOH-Aktivierung. Diese Arbeit zeigt, daß die Alkaliaktivierung des erhitzten und aufgemahlenen Meta-Illit/Smektit Tons aus Friedland unter hydrothermale Bedingungen Geopolymere mit hoher Festigkeit bildet