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G protein-coupled receptors (GPCRs) as the most important class of pharmacological targets regulate G-protein and β-arrestin-mediated signaling through allosteric interplay, which are responsible for different biochemical and physiological actions like therapeutic efficacy and side effects. However, the allosteric mechanism underlying preferentially recruiting one transducer

Although a variety of learning benefits of peer assessment have been documented, many concerns remain regarding its reliability and validity, especially for peers at early learning stages and for E...

β2AR is an important drug target protein involving many diseases. Biased drugs induce specific signaling and provide additional clinical utility to optimize β2AR-based therapies. However, the biased signaling mechanism has not been elucidated. Motivated by the issue, we chose four agonists with divergent bias (balanced agonist, G-protein-biased agonist, and β-arrestin-biased agonists) and utilized Gaussian accelerated molecular dynamics simulation coupled with a dynamic network to probe the molecular mechanisms of distinct biased activation induced by the structural differences between the four agonists. Our simulations reveal that the G-protein-biased agonist induces an open conformation with the outward shifts of TM6 and TM7 for the intracellular domain, which will be beneficial to couple G protein. In contrast, the β-arrestin-biased agonists regulate an occluded conformation with a slightly outward movement of TM6 and an inward shift of TM7, which should favor β-arrestin signaling. The balanced agonist does not induce an observable outward shift for TM6 but, along with a slight tilt for TM7, leads to an inactive-like conformation. In addition, our results reveal the first time that ICL3 presents specific conformations with different agonists. The G-protein-biased agonist drives ICL3 to open so that the G protein-binding pocket can be available, while the β-arrestin-biased agonists induce ICL3 to form a closed conformation with a stable local α-helix. MM/PBSA analysis further reveals that the hydroxyl groups in the resorcinol of the G-protein-biased agonist form strong interactions with Y5.38 and S5.42, thus preventing tilting of the TM5 extracellular end. The catechol of the balanced agonist and the β-arrestin-biased ones induces the rearrangement of two hydrophobic residues F6.52 and W6.48. However, different from the balanced agonist, the ethyl substituent of β-arrestin-biased agonists forms additional hydrophobic interactions with W6.48 and F6.51 after the rearrangement, which should contribute to the β-arrestin bias. The shortest pathway analysis further reveals that the three residues Y7.43, N7.45, and N7.49 are crucial for allosterically regulating G-protein-biased signaling, while the two residues W6.48 and F6.44 make an important contribution to regulate β-arrestin-biased signaling. For the balanced agonist NE, the allosteric regulation pathway simultaneously involves the residue associated with G-protein-biased signaling like S5.46 and the residues related to β-arrestin-biased signaling like W6.48 and F6.44, thus producing unbiased signaling. The observations could advance our understanding of the biased activation mechanism on class A GPCRs and provide a useful guideline for the design of biased drugs.

The residue located at 15 positions before the most conserved residue in TM7 (7.35 of Ballesteros-Weinstein number) plays important roles in ligand binding and the receptor activity for class A GPCRs. Nevertheless, its regulation mechanism has not been clearly clarified in experiments, and some controversies also exist for its impact on μ-opioid receptors (μOR) bound by agonists. Thus, we chose the μ-opioid receptor (μOR) of class A GPCRs as a representative and utilized a microsecond accelerated molecular dynamics simulation (aMD) coupled with a protein structure network (PSN) to explore the effect of W318sup7.35/supon its functional activity induced by the agonist endomorphin2 mainly by a comparison of the wild system and its W7.35A mutant. When endomorphin2 binds to the wild-type μOR, TM6 in μOR moves outward to form an open intracellular conformation that is beneficial to accommodating the β-arrestin transducer, rather than the G-protein transducer due to the clash with the α5 helix of G-protein, thus acting as a β-arrestin biased agonist. However, the W318A mutation induces the intracellular part of μOR to form a closed state, which disfavors coupling with either G-protein or β-arrestin. The allosteric pathway analysis further reveals that the binding of endomorphin2 to the wild-type μOR transmits more activation signals to the β-arrestin binding site while the W318A mutation induces more structural signals to transmit to common binding residues of the G protein and β-arrestin. More interestingly, the residue at the 7.35 position regulates the shortest allosteric pathway in indirect ways by influencing the interactions between other ligand-binding residues and endomorphin2. W293sup6.48/supand F289sup6.44/supare important for regulating the different activities of μOR induced either by the agonist or by the mutation. Y336sup7.53/sup, F343sup8.50/sup, and D340sup8.47/supplay crucial roles in activating the β-arrestin biased signal induced by the agonist endomorphin2, while L158sup3.43/supand V286sup6.41/supdevote important contributions to the change in the activity of endomorphin2 from the β-arrestin biased agonist to the antagonist upon the W318A mutation.

Enterovirus, like the majority of RNA viruses, evolves to survive the changeable environments by a variety of strategies. Here, we showed that HY12 virus evolved to alter its characteristics and pathogenicity by employing a non-synonymous mutation. Analyses of 5′UTR, VP1 and VP2 gene sequences revealed the existence of HY12 virus in an array of mutants defined as quasispecies. The determination of diversity and complexity showed that the mutation rate and complexity of HY12 virus quasispecies increased, while the proportion of HY12 VP1 and VP2 consensus (master) sequences decreased with increasing passages. Synonymous mutation and non-synonymous mutation analysis displayed a positive selection for HY12 quasispecies evolution. A comparison of HY12 virus in different passages demonstrated that HY12 virus altered its characteristic, phenotype, and pathogenicity via non-synonymous mutation. These findings revealed the evolution pattern for HY12 virus, and the alteration of HY12 virus characteristics and pathogenicity by mutation.

Metabotropic glutamate receptors of class C GPCRs exist as constitutive dimers, which play important roles in activating excitatory synapses of the central nervous system. However, the activation mechanism induced by agonists has not been clarified in experiments. To address the problem, we used microsecond all-atom molecular dynamics (MD) simulation couple with protein structure network (PSN) to explore the glutamate-induced activation for the mGluR1 homodimer. The results indicate that glutamate binding stabilizes not only the closure of Venus flytrap domains but also the polar interaction of LB2-LB2, in turn keeping the extracelluar domain in the active state. The activation of the extracelluar domain drives transmembrane domains (TMDs) of the two protomers closer and induces asymmetric activation for the TMD domains of the two protomers. One protomer with lower binding affinity to the agonist is activated, while the other protomer with higher binding energy is still in the inactive state. The PSN analysis identifies the allosteric regulation pathway from the ligand-binding pocket in the extracellular domain to the G-protein binding site in the intracellular TMD region and further reveals that the asymmetric activation is attributed to a combination of trans-pathway and cis-pathway regulations from two glumatates, rather than a single activation pathway. These observations could provide valuable molecular information for understanding of the structure and the implications in drug efficacy for the class C GPCR dimers.

Prostate cancer (PRAD) is a major cause of cancer-related deaths. Current monotherapies show limited efficacy due to often rapidly emerging resistance. Combination therapies could provide an alternative solution to address this problem with enhanced therapeutic effect, reduced cytotoxicity, and delayed the appearance of drug resistance. However, it is prohibitively cost and labor-intensive for the experimental approaches to pick out synergistic combinations from the millions of possibilities. Thus, it is highly desired to explore other efficient strategies to assist experimental researches. Inspired by the challenge, we construct the transcriptomics-based and network-based prediction models to quickly screen the potential drug combination for Prostate cancer, and further assess their performance by in vitro assays. The transcriptomics-based method screens nine possible combinations. However, the network-based method gives discrepancies for at least three drug pairs. Further experimental results indicate the dose-dependent effects of the three docetaxel-containing combinations, and confirm the synergistic effects of the other six combinations predicted by the transcriptomics-based model. For the network-based predictions, in vitro tests give opposite results to the two combinations (i.e. mitoxantrone-cyproheptadine and cabazitaxel-cyproheptadine). Namely, the transcriptomics-based method outperforms the network-based one for the specific disease like Prostate cancer, which provide guideline for selection of the computational methods in the drug combination screening. More importantly, six combinations (the three mitoxantrone-containing and the three cabazitaxel-containing combinations) are found to be promising candidates to synergistically conquer Prostate cancer.

Building upon Zimmerman’s socio-cognitive view of self-regulation, we explored EFL (English as a Foreign Language) students’ revision and the likely contribution to revision from three salient self-regulating sources: peer feedback, instructor feedback, and revision goals. Data was obtained from 70 Chinese EFL students in a writing class through a 300-word online writing assignment involving online instructor and peer feedback, free-response revision goals, and a required revision. We closely coded students’ revision and then used the same coding scheme to analyze the relative levels of association of revision changes with peer comments, instructor comments and revision goals. We found that: (a) the majority of revision changes have been triggered by three mediating sources, with revision goals as the most significant contributing source. Additionally, most revision changes come from a combination of two or three sources, with the overlap of peer feedback and revision goals accounting for the biggest overlapping contribution for both high and low-level revisions; (b) as for the relationship among the three sources, no significant difference was found between revision goals’ overlap rate with peer feedback and their overlap rate with instructor feedback. Instructor feedback and peer feedback did not overlap very much. Findings suggest that students could revise beyond instructor and peer feedback in their revision efforts guided by their own reflective goals, and peer feedback could function as a more productive and multiple-reader source of revision in comparison with instructor feedback. This study also provided evidence for students’ self-regulated learning of writing through the use of self-regulating resources and charted a route for how writing could be improved.

As a nutrient in body functions, folic acid (FA) plays a very important role for human health, and thus developing a highly sensitive method for its determination is of great significance. In the present work, carbon hollow nanospheres decorated with molybdenum disulfide nanosheets (CHN@MoS2) nanomaterials were produced through a simple method and adopted to modify a glassy carbon electrode for assembling a highly sensitive electrochemical sensor of FA. After characterizing the prepared nanomaterials using scanning-/transmission-electron microscopy and Raman spectra, as well as optimizing various testing conditions, including the pH value of the buffer solution, the accumulation time and amount of nanomaterials on electrode surface, and the electrochemical determination of FA was carried out using a CHN@MoS2 electrode. Owing to the coordinative advantages from CHN and MoS2, the results show that CHN@MoS2 exhibits excellent sensing responses for FA, and it has a wide linear range from 0.08 to 10.0 μM coupled with a low detection limit of 0.02 μM. Finally, the proposed method for FA detection was successfully applied in human urine analysis. The obtained results are satisfactory, revealing that the developed method based on CHN@MoS2 nanomaterials has important applications for FA determination.

In this work, accelerated molecular dynamics (aMD) simulations were used to study different effects of G286F and R126 mutations on the activity of CCR5. Potential of Mean Force (PMF) results indicate that there are stable inactive-like states and active-like ones existing in the conformation space of the wild type (WT), confirming that CCR5 could possess to some extent constitutive activity. But the R126N mutation could constrain CCR5 in the inactive state through influencing the TXP motif and limiting the movements of TM5 and TM6. In contrast, the G286F mutation promotes the activity of the receptor by increasing the distance of TM2-TM6 and the flexibility of the intracellular part of TM5 and changing the H-bonding in the TXP motif. The observations from the cross correlation analysis further show that the R126N mutation dramatically reduces the motion correlations between TMs, which should partly contribute to the deactivation of CCR5. Compared with the WT system, TM6 and TM7 in the G286F mutant are loosely correlated with other regions, which should be conducive to drive the movement of TM6 and TM7 toward the active conformation. In addition, the result from the protein structure network (PSN) analysis reveals that the shortest pathways connecting the extracellular and the intracellular domains are highly conserved in the three systems despite the different mutations, in which the hydrogen bond plays a pivotal role. However, the G286F mutation shortens the lifetime of the pathway with respect to the R126N mutation, which may be associated with the different activities of the two mutants. The pathway connecting the ligand-binding site and the G-protein region reveals that the allosteric communication between TM6 and TM7 is enhanced by the R126N mutation while the G286F mutation induces the activation of the G-protein pocket by arousing more residues in the NPxxY region to participate in the pathway.

Peer assessment has been widely applied to actively engage students in learning to write. However, sometimes students resist peer assessment. This study explores reviewers’ attitudes and other underlying factors that influence students’ participation in online peer assessment. Participants were 234 Chinese undergraduates from two different academic backgrounds: engineering majors (n = 168) and English majors (n = 66). Gender, academic background and prior experience with peer assessment were all related to participation levels. Moreover, factor analyses revealed three attitudinal factors: (1) positive attitude (a general endorsement of the benefits of peer assessment), (2) interpersonal negative (concerns about the negative effects on interpersonal relationships), and (3) procedural negative (doubts about the procedural rationality of peer assessment). Among the attitudinal factors, procedural negative was negatively associated with participation, as expected. Interestingly, interpersonal negative...

Building upon self-regulated learning theories, we examined the nature of student writing goals and the relationship of these writing goals to revision alone and in combination with two other important sources of students’ self-regulated revision—peer comments on their writing, and reflections for their own writing obtained from reviewing others’ writing. Data were obtained from a large introductory undergraduate class in the context of two 1000-word writing assignments involving online peer review and a required revision. We began with an investigation of students’ free response learning goals and a follow-up quantitative survey about the nature and structure of these writing goals. We found that: (a) students tended to create high-level substantive goals more often, (b) students change their writing goals across papers even for a very similar assignment, and (c) their writing goals divide into three dimensions: general writing goals, genre writing goals, and assignment goals. We then closely coded and analyzed the relative levels of association of revision changes with writing goals, peer comments, reflections from peer review, and combinations of these sources. Findings suggest that high-level revisions are commonly associated with writing goals, are especially likely to occur for combinations of the three sources, and peer comments alone appeared to make the largest contributions to revision.

The photothermal-chemical combination therapy is a promising approach for cancer treatment, however, chemotherapy often causes severe toxic and side effects on normal tissues. Herein, tumor-specific FeOOH@PNE-Art nanoparticles were fabricated via coating poly(norepinephrine) (PNE) on FeOOH nanoparticles, followed by loading of artemisinin (Art). The as-prepared nanoparticles exhibited excellent biocompatibility, strong near-infrared (NIR) absorbance and pH-responsive synchronous release of Art and iron ions. The released iron ions could not only supply iron ions in cancer cells which mediate endoperoxide bridge cleavage of Art and generate reactive oxygen species (ROS), but also react with H2O2 at tumour sites via the Fenton reaction and produce hydroxyl radicals, inducing a tumour-specific killing. Moreover, owing to the synchronous release of Art and iron ions as well as the low leakage of iron ions, FeOOH@PNE-Art nanoparticles showed extremely low toxicity to normal tissue. Under NIR light irradiation, the tumours in FeOOH@PNE-Art injected mice were thoroughly eliminated after 7 days of treatment and no tumour recurrence was found 30 days after treatment, manifesting very high efficacy of combination therapy.

Intracellular organelle cross-talk is a new and important research area. Under stress conditions, the coordinated action of the autophagy and endosomal systems in tumor cells is essential for maintaining cellular homeostasis and survival. The activation of the IκB kinase (IKK) complex is also involved in the regulation of stress and homeostasis in tumor cells. Here, we try to explore the effects of constitutively active IKKβ subunits (CA-IKKβ) on autophagy and endosomal system interactions. We confirm that CA-IKKβ induces accumulation of autophagosomes and their fusion with MVBs to form amphisomes in cancer cells, and also drives the release of EVs containing autophagy components through an amphisome-dependent mechanism. We further demonstrate that CA-IKKβ inhibits the expression of RAB7, thereby weakening the lysosomal-dependent degradation pathway. CA-IKKβ also induces phosphorylation of SNAP23 at Ser95 instead of Ser110, which further promotes amphisome-plasma membrane fusion and sEV secretion. These results indicate that CA-IKKβ drives the formation and transport of amphisomes, thereby regulating tumor cell homeostasis, which may illuminate a special survival mechanism in tumor cells under stress.

Glucagon-like peptide-1 receptor (GLP-1R), as a member of the class B G protein-coupled receptors (GPCRs), plays a crucial role in regulating blood glucose level signal recognition through its activation. The conformation changes during the activation pathway are of particular importance for its function. To investigate the activation mechanism of GLP-1R, the crystal structures of active and inactive forms are chosen to perform a total of 2 μs of accelerated molecular dynamics (aMD) simulations and 400ns of conventional molecular dynamics (cMD) simulations. With the aid of structural analysis and potential of mean force (PMF) calculations, we reveal the role of different helices in the activation and deactivation process and obtain the intermediate states during activation and deactivation that are difficult to capture in experiments. Protein structure network (PSN) was utilised to clarify the allosteric communication pathways of activation and deactivation and reveal the mechanisms of its activation and deactivation. The results could advance our understanding of the activation mechanism of GLP-1R and the related drug design.