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Gold(I)-catalyzed stereoselective β-glycosylation of primary alcohols is achieved using the orthogonally protected mannosyl α

The bacteria-derived formyl peptide fMet-Leu-Phe (fMLF) is a potent chemoattractant of phagocytes that induces chemotaxis at subnanomolar concentrations. At higher concentrations, fMLF inhibits chemotaxis while stimulating degranulation and superoxide production, allowing phagocytes to kill invading bacteria. How an agonist activates distinct cellular functions at different concentrations remains unclear. Using a bioluminescence resonance energy transfer–based FPR1 biosensor, we found that fMLF at subnanomolar and micromolar concentrations induced distinct conformational changes in FPR1, a Gi-coupled chemoattractant receptor that activates various phagocyte functions. Neutrophil-like HL-60 cells exposed to subnanomolar concentrations of fMLF polarized rapidly and migrated along a chemoattractant concentration gradient. These cells also developed an intracellular Ca2+concentration gradient. In comparison, high nanomolar and micromolar concentrations of fMLF triggered the PLC-β/diacyl glycerol/inositol trisphosphate pathway downstream of the heterotrimeric Gi proteins, leading to Ca2+mobilization from intracellular stores and Ca2+influx from extracellular milieu. A robust and uniform rise in cytoplasmic Ca2+level was required for degranulation and superoxide production but disrupted cytoplasmic Ca2+concentration gradient and inhibited chemotaxis. In addition, elevated ERK1/2 phosphorylation and β-arrestin2 membrane translocation were associated with diminished chemotaxis in the presence of fMLF above 1 nM. These findings suggest a mechanism for FPR1 agonist concentration–dependent signaling that leads to a switch from migration to bactericidal activities in phagocytes.

In this letter, an 8x8 multiple-input-multiple-output (MIMO) antenna that operates in the sub-6 GHz (4.7-5.1 GHz) spectrum for 5G MIMO smartphone applications is presented. The design consists of a fully grounded plane with closely orthogonal antenna pairs placed symmetrically on the corners of the smartphone and each antenna element consists of an F-type monopole with dimensions of 18x7.5mm2. Firstly, the diversity characteristics of orthogonally placed antennas are used to reduce the coupling between antennas. On the other hand, diagonal antennas are connected by a neutral line (NL) to further improve isolation and etched slots on the ground to reduce the coupling between antenna pairs. A good frequency bandwidth (S11 < -10 dB) of 4.7-5.1 GHz has been obtained for the MIMO antenna, and the isolation is lower than 16 dB. In addition, total efficiency (TE) is greater than 77.5%, and the envelope correlation coefficient (ECC) is lower than 0.025. The fabricated antenna prototype is tested and offers good performance.

G-Protein-coupled receptors (GPCRs) belong to an important family of integral membrane receptor proteins that are essential for a variety of transmembrane signaling process, such as vision, olfaction, and hormone responses. They are also involved in many human diseases (Alzheimer's, heart diseases, etc.) and are therefore common drug targets. Thus, understanding the details of the GPCR activation process is a task of major importance. Various types of crystal structures of GPCRs have been solved either at stable end-point states or at possible intermediate states. However, the detailed mechanism of the activation process is still poorly understood. For example, it is not completely clear when the nucleotide release from the G protein occurs and how the key residues on α5 contribute to the coupling process and further affect the binding specificity. In this work we show by free energy analysis that the guanosine diphosphate (GDP) molecule could be released from the Gs protein when the binding cavity is half open. This occurs during the transition to the Gs open state, which is the rate-determining step in the system conformational change. We also account for the experimentally observed slow-down effects by the change of the reaction barriers after mutations. Furthermore, we identify potential key residues on α5 and validated their significance by site-directed mutagenesis, which illustrates that computational works have predictive value even for complex biophysical systems. The methodology of the current work may be applied to other biophysical systems of interest.

“Student-centered” is the essential requirement of higher education in the new era, and the inevitable requirement of the reform of higher education talent cultivation mode, as well as the starting point and end-result of the innovation of teaching mode. College of Electronic Information Engineering of Inner Mongolia University takes the opportunity of Undergraduate Engineering Education Accreditation and Undergraduate Professional Certification to explore the “student-centered” talent cultivation mode, building “students as the center, learning + teaching + innovative practice experience” ternary supporting talent cultivation mode. To be specific, “student-centered” innovation talent cultivation as the main line, “virtue and goodness” and “dedication to learning and innovation” as the cultivation objective, cultivating students’ knowledge, ability and quality as the fundamental, three measures are adopted to cultivate high-quality specialized technical talents. The measures mainly include building a diversified cultivation system to promote the transformation of students’ learning mode, deepening the reform of curriculum structure to promote the transformation of teachers’ teaching mode, building a platform for entrepreneurship and innovation education to enhance students’ practical experience. Practice shows that the mode can help students form comprehensive literacy to solve practical problems, forward-looking professional vision and autonomous lifelong learning ability.

Perpendicular shape anisotropy spin-transfer-torque magnetic random-access memory (PSA-STT-MRAM) demonstrates high thermal stability when size is reduced to 20 nm, which gives a new way to improve the integrity of electronic devices. This long and narrow device also poses challenges in the device fabrication process, such as sample tilt and etching defects. We used a micromagnetic simulation method to investigate the relationship between those defects and device performance. The coercivity and critical switching current density of PSA-MRAM have been calculated and analyzed with micromagnetic simulation, a three-dimensional Stoner–Wohlfarth model, and spin-filter theory. Our results demonstrate how shape defects affect the performance of the PSA-MRAM and provide guidelines for practical realization of nanoscale PSA-MRAM.

The reform of any course cannot be accomplished in one move and needs to be improved continuously for a long time. The scheme of blended learning reforms was shown from selecting online learning resources, dividing curriculum content, the problem of Blending learning hours and the implementation of discussion and cooperative learning in this paper. We also discussed some problems faced by Fundamentals of Circuit Analysis course in the Blending learning reform, and shared some of the accumulated experience and relevant suggestions. After 4 years’ blended learning reforms, the class based on Fundamentals of Circuit Analysis has effectively improved the dull state, and enhanced students’ interest in learning, which has also significantly improved the teaching effect. At the same time, this “student-centered” teaching design plays a great role in cultivating students’ innovation ability.

While conventional microelectronic integrated circuits based on electron charges approach the theoretical limitations in foreseeable future, next-generation nonvolatile logic units based on electro...

This paper proposes a multifunctional metamaterial device operating in the terahertz (THz) band. The metamaterial device can switch functions by using the phase transition properties of vanadium dioxide (VO2) and the photoconductive effect of silicon. An intermediate metal layer divides the device into the I side and II side. When VO2 is in the insulating state, the I side can achieve polarization conversion from linear polarization waves to linear polarization waves at 0.408–0.970 THz. When VO2 is in the metal-like state, the I side can perform polarization conversion from linear polarization waves to circular polarization waves at 0.469–1.127 THz. When silicon is not excited in the absence of light, the II side can perform polarization conversion from linear polarization waves to linear polarization waves at 0.799–1.336 THz. As the light intensity increases, the II side can realize stable broadband absorption at 0.697–1.483 THz when silicon is in the conductive state. The device can be applied to wireless communications, electromagnetic stealth, THz modulation, THz sensing, and THz imaging. Moreover, it provides a fresh idea for the design of multifunctional metamaterial devices.

Objectives We engineered a CRISPR interference (CRISPRi) system targeting the AcrAB-TolC efflux pump to prevent MDR development in Escherichia coli. Methods Nine specific single-guide RNAs (sgRNAs) were designed to target the components of the AcrAB-TolC efflux pump, namely AcrA, AcrB and TolC. A total of thirteen CRISPRi recombinant plasmids were constructed with single or clustered sgRNAs. The transcriptional levels of the target genes, MICs of multiple antibiotics and biofilm formation in each CRISPRi strain were tested. Results The CRISPRi system expressing sgRNA clusters targeting acrB and tolC simultaneously exhibited the highest inhibitory effect on AcrAB-TolC efflux pump activity in E. coli HB101, with 78.3%, 90.0% and 65.4% inhibition rates on the transcriptional levels of acrA, acrB and tolC, respectively. The CRISPRi system resulted in ∼2-, ∼8- and 16-fold increased susceptibility to rifampicin, erythromycin and tetracycline, respectively. In addition, the constructed CRISPRi system reduced biofilm formation with inhibition rates in the range of 11.2% to 58.2%. Conclusions To the best of our knowledge, this is the first report on the construction of an inducible CRISPRi system targeting the AcrAB-TolC efflux pump to prevent MDR development in E. coli. This study provides insights for future regulation and manipulation of AcrAB-TolC activity and bacterial MDR by a CRISPRi system.

Objectives Polycyclic aromatic hydrocarbons (PAHs) and 3-monochloropropane-1,2-diol ester (3-MCPDE) were studied in camellia oil. It is important to study the changes in the content of PAHs and 3-MCPDE at different refining stages (from crude oil to the final refined oil product) to elucidate the influence of the refining procedures on their change. Materials and methods The PAHs and 3-MCPDE in camellia oil from different refining stages (from crude oil to the product) of a plant were analyzed by gas chromatography–mass spectrometry and calculated by the internal standard method. Results The overall PAH content was (79.64±2.43) µg/kg in crude camellia oil. After refining treatment, the PAH content decreased to (18.75±0.55) µg/kg. The 3-MCPDE content increased during the refining process from 0 mg/kg in the crude oil to 4.62 mg/kg in the refined oil product. Conclusions This is the first study to simultaneously monitor changes in both PAH and 3-MCPDE contents during the production of camellia oil. These results confirmed the effectiveness of the refining method on PAH removal and the increase in 3-MCPDE at high temperature. It is suggested that novel processing methods or refining parameters need further optimization to decrease the overall concentrations of PAHs and 3-MCPDE in camellia oil.

A gas chromatography-mass spectrometry(GC-MS) method for the determination of 16 European priority polycyclic aromatic hydrocarbons(16 EUPAHs) in infant formula milk powder was established, and the characterization and investigation of 16 EUPAHs in 70 milk formula powders were carried out in 2020.After hydrolysis, extraction, saponification and solid phase extraction, infant formula milk powder was detected by GC-MS using DB-EUPAH capillary column(20 m×0.18 mm, 0.14 μm)and quantified by internal standard method.The average recoveries ranged from 67.8% to 116.2% and the relative standard deviations ranged from 2.0% to 15.1%(n=6). The limits of quantification and detection of the method were 0.5 and 0.2 μg/kg, respectively. The content of 16 EUPAHs waslt;0.2-0.48 μg/kg, including PAH4 content in the range oflt;0.2-0.91 μg/kg, the characterization and investigation of infant formula powder in 16 EUPAHs mainly chrysene, cyclopenta[c, d]pyrene, benz[a]anthracene, benzo[b]fluoranthene, benzo[g, h, i]perylene.The method is simple, accurate and suitable for the determination of 16 EUPAHs in infant formula milk powder. The result showed that the content of 16 EUPAHs in commercially available infant formula milk powder in Hangzhou was low and all of them met the limit requirement of European Union.

Protein lysine acetylation is an evolutionarily conserved post-translational modification (PTM), which is dynamic and reversible, playing a crucial regulatory role in almost every aspect of metabolism, of both eukaryotes and prokaryotes. Several global lysine acetylome studies have been carried out in various bacteria, but thus far, there have been no reports of lysine acetylation for the commercially important aquatic animal pathogen

Frequency selective surfaces (FSSs), modern artificial materials, show great potential in engineering applications due to their excellent frequency selection capabilities. In this paper, we introduce a flexible strain sensor based on FSS reflection characteristics, which can be well conformally attached to the surface of an object and bear mechanical deformation from a certain load. When the FSS structure changes, the original working frequency will be shifted. By measuring the difference in electromagnetic performance, the strain degree of the object can be monitored in real-time. In this study, we designed an FSS sensor with a working frequency of 31.4 GHz and amplitude that reaches -35 dB that exhibits favorable resonance properties in the Ka-band. The quality factor of FSS is 16.2, which indicates that the sensor has excellent sensing performance. The sensor was applied in the strain detection of a rocket engine case through statics and electromagnetic simulations. The analysis showed that the working frequency of the sensor shifted by approximately 200 MHz for 1.64% radial expansion of the engine case and the frequency shift exhibits an excellent linear relationship with the deformation in diverse loads, so it can be used for accurate strain detection of the case. Based on experiments, we carried out the uniaxial tensile test of the FSS sensor in this study. The sensor’s sensitivity was 1.28 GHz/mm when the FSS was stretched by 0–3 mm in the test. Therefore, the FSS sensor has high sensitivity and strong mechanical properties, which verifies the practical value of the FSS structure designed in this paper. It has a broad development space in this field.

The magnetic bimeron, as the in-plane counterpart of the magnetic skyrmion, has potential applications in next-generation spin memory devices due to its lower energy consumption. In this work, the dynamic behavior of a current-driven bimeron in a nanotrack with voltage-controlled magnetic anisotropy (VCMA) is investigated. By adjusting the profile of the VCMA, the bimeron can display a diode-like unidirectional behavior in the nanotrack. The unidirectional behavior can be modulated by changing the driven current density and width of the VCMA region. The trajectory of the bimeron can also be controlled by the periodic VCMA region, which can enhance the stability of bimeron and realize a high-storage density bimeron-based information channel.

This work aimed to establish a novel determination method for acrylamide in coffee and its products by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Acrylamide in samples were prepared by a single-step solid-phase extraction clean-up using mixed mode sorbents. The bromine derivatization efficiency of acrylamide and its internal standard were improved at an acidic condition. After derivation, the retention capability of acrylamide and its resistance to interference were significantly improved. The limit of detection (LOD) and the limit of quantification (LOQ) were 1.2 and 4 μg/kg for roasted and instant coffees, while they were 0.24 and 0.8 μg/kg for ready-to-drink coffees. The average recoveries for acrylamide ranged from 99.3 to 102.2% in coffee and its products. All the results showed that the developed method was simple, quick, specific and suitable for screening and determination of acrylamide in batch samples of coffee and its products.

The pandemic caused by SARS-CoV-2 has cost millions of lives and tremendous social/financial loss. The virus continues to evolve and mutate. In particular, the recently emerged "UK", "South Africa", and Delta variants show higher infectivity and spreading speed. Thus, the relationship between the mutations of certain amino acids and the spreading speed of the virus is a problem of great importance. In this respect, understanding the mutational mechanism is crucial for surveillance and prediction of future mutations as well as antibody/vaccine development. In this work, we used a coarse-grained model (that was used previously in predicting the importance of mutations of N501) to calculate the free energy change of various types of single-site or combined-site mutations. This was done for the UK, South Africa, and Delta mutants. We investigated the underlying mechanisms of the binding affinity changes for mutations at different spike protein domains of SARS-CoV-2 and provided the energy basis for the resistance of the E484 mutant to the antibody m396. Other potential mutation sites were also predicted. Furthermore, the in silico predictions were assessed by functional experiments. The results establish that the faster spreading of recently observed mutants is strongly correlated with the binding-affinity enhancement between virus and human receptor as well as with the reduction of the binding to the m396 antibody. Significantly, the current approach offers a way to predict new variants and to assess the effectiveness of different antibodies toward such variants.

We present in this paper an extension of the guided-wave M-component model of Rakov et al. considering the presence of a vertically elevated strike object. The tall object is represented as a lossless, uniform transmission line. Expressions for the current distribution along the channel and along the strike object are derived. Simulation results for the electric field at close (100 m), intermediate (15 km), and far (100 km) distance ranges are presented, considering fast and slow M-component currents. The results show that, for very tall structures and fast M-component waves, the presence of a tall strike object can result in a sharp peak superimposed on the M-component electric field at intermediate and far distance ranges (e.g., 15 and 100 km). At close distances, the electric field of a fast M-component is characterized by an initial negative excursion followed by a polarity change. It is shown that the presence of the tower results in a decrease of the negative initial excursion of the field. For slow M-component waveforms or for moderately tall structures, the presence of the tall strike object can be disregarded in the M-component field calculations. A discussion on the influence of the M-wave channel height and velocity on the electric fields for fast M-component currents is also presented. The study suggests a noticeable effect of the M-wave velocity on the electric fields at all distance ranges. The variations of the wave velocity and channel length affect the time of appearance of the superimposed sharp peak on the electric fields. However, the sharp peak magnitude, which is due to the presence of the tower, is independent of the channel length and wave velocity. It is determined by the injected current and the reflection coefficients from the ground and tower top.

The survival and immune responses of Litopenaeus vannamei were evaluated during white spot syndrome virus (WSSV) or Vibrio parahaemolyticus single and concurrent infections. The mortality, WSSV load, activities of 4 immune enzymes: acid phosphatase (ACP), alkaline phosphatase (AKP), peroxidase (POD) and superoxide dismutase (SOD), and the transcription of Evolutionarily Conserved Signaling Intermediate in Toll pathways of L.vannamei (LvECSIT) were quantified at 0, 3, 6, 12, 24, 48, 72 and 96 h post-infection (pi). The results showed: (i) the cumulative mortality of the co-infection group (WSSV and V. Parahaemolyticus 83%) was significantly lower than the WSSV infection group (97%) (P < 0.05) at 96 hpi; (ii) copies of WSSV in the co-infection group were significantly lower than that of the single infection group from 24 to 96 hpi (P < 0.05); (iii) ACP, AKP,POD and SOD activity in the gills of the co-infection group was higher than that of the WSSV group at12, 48 and 96 hpi (P < 0.05).The expression of LvECSIT mRNA in the co-infection group was significantly higher than in the WSSV infection group from 12 to 72 hpi (P < 0.05).The results indicate that proliferation of WSSV is inhibited by V.parahaemolyticus infection. In addition, infection with WSSV alone causes a significant reduction in some immune responses of shrimp than co-infection with WSSV and V.parahaemolyticus occurs at 26 °C. Third, LvECSIT, an essential member of TLR signaling pathway might play a crucial role in shrimp defense against WSSV – Vibrio co-infection.

Isobutane alkylation with 2-butene to produce high-quality gasoline was catalyzed by N-methylacetamide (NMA)–AlCl3 based ionic liquid (IL) analog with a NMA/AlCl3 molar ratio of 0.75 and CuCl modification, which was marked as CuCl-modified 0.75NMA–1.0AlCl3. The long-term experiment was carried out in the autoclave operated in continuous mode to investigate the distribution of alkylate under different experimental nodes. The result indicated that the long-term alkylation was divided into three stages: rising, stable, and descending regions. C8 selectivity and molar ratio of trimethylpentanes (TMPs) to dimethylhexanes (DMHs) reached the highest level in the stable region, and research octane number (RON) of alkylate was as high as 97. Anionic Al species ([Al2Cl7]−, [AlCuCl5]−) and cationic Al species ([AlCl2L]+) from IL analog as two active Lewis acidic species played a catalytic role in the long-term alkylation, whereas the neutral Al species did not participate into the alkylation. Moreover, the structure of CuCl-modified 0.75NMA–1.0AlCl3 was destroyed after the deactivation, and CuCl was enriched in the CD2Cl2-insoluble substance, resulting in a decreasing TMP/DMH ratio. The catalytic lifetime of IL analog was similar with CuCl-modified 0.55Et3NHCl–1.0AlCl3 IL, but IL analog had a lower cost.

Ultrasound can be considered a mechanical wave for both clinical diagnostic and therapeutic purposes on the basis of its good penetrability and directivity while spreading in solid organs or tissues without any ionizing radiation. As a powerful form of energy, ultrasound, is used for deep-tissue therapy with different sonication parameters. The feasibility of minimally invasive or noninvasive acoustic treatment of a variety of diseases, such as hypertension, arrhythmia, hypertrophic cardiomyopathy, and myocardial infraction, is being explored in animal experiments and clinical trials. In this review, we summarize the biomedical effects of acoustic intervention in experimental and clinical studies, current challenges, and the potential of ultrasound for cardiovascular disease therapy.

G protein-coupled receptors (GPCRs) undergo ligand-induced internalization that carries the cognate ligands into intracellular compartments. The present study explores this property for the use of formyl peptide receptor 1 (FPR1), a class A GPCR that binds formylated peptides, as a potential target for drug delivery. A pH-sensitive peptide-drug conjugate consisting of doxorubicin (DOX), N-e-maleimidocaproic acid hydrazide (EMCH), and the formyl peptide fMet-Leu-Phe-Cys (abbreviated as DEF) was prepared. DEF retained pharmacological activities of formyl peptides in binding to FPR1 and mobilization of Ca2+ from intracellular stores. However, the conjugated DOX was no longer cell membrane-permeable and relied on FPR1 for cellular entry. DOX was released from DEF into acidic compartments labeled with fluorescent trackers for endosomes. Treatment of cells with pharmacological inhibitors that block clathrin- or caveolae-mediated endocytosis did not abrogate FPR1-dependent DEF internalization, nor did inhibition of macropinocytosis and phagocytosis. In contrast, cholesterol depletion abrogated DEF internalization through FPR1, suggesting characteristics of cholesterol-dependent uptake mediated by a cell surface receptor. These results demonstrate the possibility of using FPR1 for targeted drug delivery.

Active ingredients from Chinese medicine (AIFCM) are emerging as a class of anticancer agents for treating hepatocellular carcinoma (HCC). AIFCM have unique advantages over traditional chemotherapeutic drugs, such as their multi-target, multi-level, and coordinated intervention effects against HCC. Recent researches demonstrated their potentials in inhibiting HCC initiation, cell proliferation, angiogenesis, and metastasis. With the advantages of nano-carriers including better bioavailability, significant features of passive or active targeting and stimulus-responsive drug release. Formulation of AIFCM and nano-carriers shows great potential in promoting anti-HCC therapeutic development. Here, we critically assess nano-carriers for delivery and targeting of AIFCM for HCC therapy. First, we offer an overview of major pathogenic factors to induce HCC, including virus infection, alcoholic liver disease, mycotoxin, obesity, and type 2 diabetes. Second, the mechanisms of AIFCM for HCC therapy comprising gene regulation and transduction of different molecular pathways are discussed. Third, significant achievements of AIFCM-encapsulated nano-carriers and AIFCM-modified nano-carriers against HCC are presented. Finally, we provide concluding remarks and future perspectives of AIFCM for HCC therapy, hoping to offer some insight on how to improve AIFCM-based HCC therapy.

Funding Information: The atomistic simulations were undertaken on the VIKING cluster, which is a high performance computing facility provided by the University of York. We are grateful for computational support from the University of York High Performance Computing service, VIKING, and the Research Computing team. The work of A.D. and L.S. was supported by the Ministry of Innovation and Technology and the National Research, Development and Innovation Office under Projects No. PD134579 and No. K131938, as well as within the Quantum Information National Laboratory of Hungary. Publisher Copyright: © 2021 American Physical Society. Skyrmions are topologically protected nanoscale magnetic structures with a wide range of potential applications. Here we determine the life cycle of skyrmions from their creation to their intrinsic dynamics and thermal stability to their eventual thermodynamic demise. Using atomistic simulations of Ir/Co/Pt, parameterized from ab initio calculations, we demonstrate the thermal phase transition to a skyrmion state under application of a perpendicular magnetic field. The created skyrmions exhibit Brownian particlelike dynamics driven by the underlying thermal spin fluctuations. At an elevated temperature window well below the Curie temperature, the skyrmions are metastable and can collapse to a uniform magnetic state. With application of an external magnetic field, the intrinsic local thermal spin fluctuations at this elevated temperature window are sufficiently large to allow the spontaneous formation of new skyrmions in thermodynamic equilibrium analogous to a spontaneous skyrmion gas. Such a system could be used to implement a skyrmion-based true random number generator.

To explore the genetic basis for a Chinese pedigree affected with genetic epilepsy with febrile seizures plus (GEFS+).Clinical data of the proband and his family members were collected. Following extraction of genomic DNA, the proband was subjected to high-throughput sequencing. Candidate variant was verified by Sanger sequencing of the proband and other family members.The pedigree, including 6 patients with febrile seizures from 3 generations, was diagnosed with typical GEFS+. Among them, 2 had febrile seizures (FS), 1 had febrile seizures plus (FS+), and 3 had febrile seizures with focal seizures. High-throughput sequencing revealed that the proband has carried a heterozygous missense variant of c.4522TA (p.Tyr1508Asn) of the SCN1A gene. Sanger sequencing confirmed that other five patients and one normal member from the pedigree have also carried the same variant, which yielded a penetrance of 85.7%.The c.4522TA (p.Tyr1508Asn) of the SCN1A gene probably underlay the disease in this pedigree. The pattern of inheritance was consistent with autosomal dominant inheritance with incomplete penetrance. Above finding has enriched the variant spectrum of the SCN1A gene.

G-Protein-coupled receptors (GPCRs) belong to an important family of integral membrane receptor proteins that are essential for a variety of transmembrane signaling process, such as vision, olfaction, and hormone responses. They are also involved in many human diseases (Alzheimer's, heart diseases, etc.) and are therefore common drug targets. Thus, understanding the details of the GPCR activation process is a task of major importance. Various types of crystal structures of GPCRs have been solved either at stable end-point states or at possible intermediate states. However, the detailed mechanism of the activation process is still poorly understood. For example, it is not completely clear when the nucleotide release from the G protein occurs and how the key residues on α5 contribute to the coupling process and further affect the binding specificity. In this work we show by free energy analysis that the guanosine diphosphate (GDP) molecule could be released from the G