Engineering APOBEC3A deaminase for highly accurate and efficient base editing.

Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

Subtle Structural Changes across the Boundary between A2AR/A2BR Dual Antagonism and A2BR Antagonism: A Novel Class of 2-Aminopyrimidine-Based Derivatives.

Aberrantly elevated adenosine in the tumor microenvironment exerts its immunosuppressive functions through adenosine receptors A2AR and A2BR. Antagonism of A2AR and A2BR has the potential to suppress tumor growth. Herein, we report a systemic assessment of the effects of an indole modification at position 4, 5, 6, or 7 on both A2AR/A2BR activity and selectivity of novel 2-aminopyrimidine compounds. Substituting indole at the 4-/5-position produced potent A2AR/A2BR dual antagonism, whereas the 6-position of indole substitution gave highly selective A2BR antagonism. Molecular dynamics simulation showed that the 5-cyano compound 7ai had a lower binding free energy than the 6-cyano compound 7aj due to water-bridged hydrogen bond interactions with E169 or F168 in A2AR. Of note, dual A2AR/A2BR antagonism by compound 7ai can profoundly promote the activation and cytotoxic function of T cells. This work provided a strategy for obtaining novel dual A2AR/A2BR or A2BR antagonists by fine-tuning structural modification.

Kisspeptin-10 binding to Gpr54 in osteoclasts prevents bone loss by activating Dusp18-mediated dephosphorylation of Src.

Osteoclasts are over-activated as we age, which results in bone loss. Src deficiency in mice leads to severe osteopetrosis due to a functional defect in osteoclasts, indicating that Src function is essential in osteoclasts. G-protein-coupled receptors (GPCRs) are the targets for ∼35% of approved drugs but it is still unclear how GPCRs regulate Src kinase activity. Here, we reveal that GPR54 activation by its natural ligand Kisspeptin-10 (Kp-10) causes Dusp18 to dephosphorylate Src at Tyr 416. Mechanistically, Gpr54 recruits both active Src and the Dusp18 phosphatase at its proline/arginine-rich motif in its C terminus. We show that Kp-10 binding to Gpr54 leads to the up-regulation of Dusp18. Kiss1, Gpr54 and Dusp18 knockout mice all exhibit osteoclast hyperactivation and bone loss, and Kp-10 abrogated bone loss by suppressing osteoclast activity in vivo. Therefore, Kp-10/Gpr54 is a promising therapeutic target to abrogate bone resorption by Dusp18-mediated Src dephosphorylation.

Structural insight into the dual-antagonistic mechanism of AB928 on adenosine A2 receptors.

The adenosine subfamily G protein-coupled receptors A2AR and A2BR have been identified as promising cancer immunotherapy candidates. One of the A2AR/A2BR dual antagonists, AB928, has progressed to a phase II clinical trial to treat rectal cancer. However, the precise mechanism underlying its dual-antagonistic properties remains elusive. Herein, we report crystal structures of the A2AR complexed with AB928 and a selective A2AR antagonist 2-118. The structures revealed a common binding mode on A2AR, wherein the ligands established extensive interactions with residues from the orthosteric and secondary pockets. In contrast, the cAMP assay and A2AR and A2BR molecular dynamics simulations indicated that the ligands adopted distinct binding modes on A2BR. Detailed analysis of their chemical structures suggested that AB928 readily adapted to the A2BR pocket, while 2-118 did not due to intrinsic differences. This disparity potentially accounted for the difference in inhibitory efficacy between A2BR and A2AR. This study serves as a valuable structural template for the future development of selective or dual inhibitors targeting A2AR/A2BR for cancer therapy.

Structural insight into CD93 recognition by IGFBP7.

The CD93/IGFBP7 axis proteins are key factors expressed in endothelial cells (EC) that mediate EC angiogenesis and migration. Their upregulation contributes to tumor vascular abnormality and a blockade of this interaction promotes a favorable tumor microenvironment for therapeutic interventions. However, the interactions of these proteins with each other remain unclear. In this study, we determined a partial structure of the human CD93-IGFBP7 complex comprising the EGF1 domain of CD93 and the IB domain of IGFBP7. Mutagenesis studies confirmed interactions and specificities. Cellular and mouse tumor studies demonstrated the physiological relevance of the CD93-IGFBP7 interaction in EC angiogenesis. Our study provides leads for the development of therapeutic agents to precisely disrupt unwanted CD93-IGFBP7 signaling in the tumor microenvironment. Additionally, analysis of the CD93 full-length architecture provides insights into how CD93 protrudes on the cell surface and forms a flexible platform for binding to IGFBP7 and other ligands.

Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos.

Base editors have substantial promise in basic research and as therapeutic agents for the correction of pathogenic mutations. The development of adenine transversion editors has posed a particular challenge. Here we report a class of base editors that enable efficient adenine transversion, including precise A•T-to-C•G editing. We found that a fusion of mouse alkyladenine DNA glycosylase (mAAG) with nickase Cas9 and deaminase TadA-8e catalyzed adenosine transversion in specific sequence contexts. Laboratory evolution of mAAG significantly increased A-to-C/T conversion efficiency up to 73% and expanded the targeting scope. Further engineering yielded adenine-to-cytosine base editors (ACBEs), including a high-accuracy ACBE-Q variant, that precisely install A-to-C transversions with minimal Cas9-independent off-targeting effects. ACBEs mediated high-efficiency installation or correction of five pathogenic mutations in mouse embryos and human cell lines. Founder mice showed 44-56% average A-to-C edits and allelic frequencies of up to 100%. Adenosine transversion editors substantially expand the capabilities and possible applications of base editing technology.

Transferrin receptor levels and its rare variant are associated with human obesity.

AIM: Iron homeostasis is critical for functional respiratory chain complex of mitochondrial, thus potentially contributing to fat biology and energy homeostasis. Transferrin receptor (Tfrc) binds to transferrin for extracellular iron uptake and is recently reported to be involved in brown fat development and functionality. However, whether TFRC levels and variants are associated with human obesity is unknown. METHODS: To investigate the association of TFRC levels and variants with human obesity, fat biopsies were obtained from surgery. Exon-sequencing and genetic assessments were conducted of a case-control study. For TFRC levels assessment in fat biopsy, 9 overweight and 12 lean subjects were involved. For genetic study, obese (n = 1271) and lean subjects (n = 1455) were involved. TFRC levels were compared in abdominal mesenteric fat of pheochromocytoma patients versus control subjects, and overweight versus lean subjects. For genetic study, whole-exome sequencing of obese and matched control subjects were conducted and analyzed. In addition, the possible disruption in protein stability of TFRC variant was assessed by structural and molecular analysis. RESULTS: TFRC levels are increased in human browning adipose tissue and decreased in fat of overweight patients. Besides, TFRC levels are negatively correlated with body mass index and positively correlated with uncoupling protein 1 levels. Furthermore, a rare heterozygous missense variant p.I337V in TFRC shows a tendency to enrich in obese subjects. Structural and functional study reveals impaired protein stability of the TFRC variant compared to wild-type. CONCLUSIONS: Reduced TFRC levels and its rare variant p.I337V with protein instability are associated with human obesity.

Structural insight into CD93 recognition by IGFBP7.

The CD93/IGFBP7 axis are key factors expressed in endothelial cells (EC) that mediate EC angiogenesis and migration. Upregulation of them contributes to tumor vascular abnormality and blockade of this interaction promotes a favorable tumor microenvironment for therapeutic interventions. However, how these two proteins associated to each other remains unclear. In this study, we solved the human CD93-IGFBP7 complex structure to elucidate the interaction between the EGF 1 domain of CD93 and the IB domain of IGFBP7. Mutagenesis studies confirmed the binding interactions and specificities. Cellular and mouse tumor studies demonstrated the physiological relevance of the CD93-IGFBP7 interaction in EC angiogenesis. Our study provides hints for development of therapeutic agents to precisely disrupt unwanted CD93-IGFBP7 signaling in the tumor microenvironment. Additionally, analysis of the CD93 full-length architecture provides insights into how CD93 protrudes on the cell surface and forms a flexible platform for binding to IGFBP7 and other ligands.

Human adenovirus type 7 subunit vaccine induces dendritic cell maturation through the TLR4/NF-κB pathway is highly immunogenic.

Introduction: Human adenovirus type 7 (HAdv-7) infection is the main cause of upper respiratory tract infection, bronchitis and pneumonia in children. At present, there are no anti- adenovirus drugs or preventive vaccines in the market. Therefore, it is necessary to develop a safe and effective anti-adenovirus type 7 vaccine. Methods: In this study, In this study, we used the baculovirus-insect cell expression system to design a recombinant subunit vaccine expressing adenovirus type 7 hexon protein (rBV-hexon) to induce high-level humoral and cellular immune responses. To evaluate the effectiveness of the vaccine, we first detected the expression of molecular markers on the surface of antigen presenting cells and the secretion of proinflammatory cytokines in vitro. We then measured the levels of neutralizing antibodies and T cell activation in vivo. Results: The results showed that the rBV-hexon recombinant subunit vaccine could promote DC maturation and improve its antigen uptake capability, including the TLR4/NF-κB pathway which upregulated the expression of MHCI, CD80, CD86 and cytokines. The vaccine also triggered a strong neutralizing antibody and cellular immune response, and activated T lymphocytes. Discussion: Therefore, the recombinant subunit vaccine rBV-hexon promoted promotes humoral and cellular immune responses, thereby has the potential to become a vaccine against HAdv-7.

Inhibitory Effects of Esculetin on Liver Cancer Through Triggering NCOA4 Pathway-Mediation Ferritinophagy in vivo and in vitro.

Objective: To explore the effects of Esculetin on liver cancer and explore potential mechanisms of Esculetin-inducing cells death. Methods: Esculetin's effects on the proliferation, migration and apoptosis of HUH7 and HCCLM3 cells were detected by using CCK8, crystal violet staining, wound healing, TranswellTM and Annexin V-FITC/PI. Flow cytometry, fluorescence staining, Western blot, T-AOC, DPPH radical scavenging assay, hydroxyl radical's inhibitory capability and GSH test were used to examine the esculetin's effects on the ROS level, the oxidation-related substances and proteins' expression in hepatoma cells. In vivo experiment was performed by xenograft model. Ferrostatin-1 was used to determine the death way of hepatoma cells induced by esculetin. Live cell probe, Western blot, Fe2+ content, MDA, HE staining, Prussian blue staining and immunohistochemistry were used to examine the ferritinophagy-related phenomenon induced by esculetin in hepatoma cells. The relationship between esculetin and NCOA4-mediated ferritinophagy was confirmed through gene silence and overexpression, immunofluorescence staining and Western blot. Results: Esculetin suppressed the proliferation, migration and apoptosis of HUH7 and HCCLM3 cells significantly, influenced the oxidative stress level, altered the autophagy and iron metabolism levels in cells, and produced a ferritinophagy-related phenomena. Esculetin increased the levels of cellular lipid peroxidation and reactive oxygen species. In vivo, esculetin could decrease tumour volume, promote LC3 and NCOA4 expressions, suppresse hydroxyl radical's inhibiting capacity and GSH, increase Fe2+ and MDA levels, decrease antioxidant proteins expression in tumour tissue. In addition, Esculetin could also increase the iron deposition of tumour tissues, promote ferritinophagy, and induce tumours' ferroptosis. Conclusion: Esculetin has an inhibitory effect on liver cancer in vivo and in vitro through triggering NCOA4 pathway-mediation ferritinophagy.

Fish gelatin films incorporated with cinnamaldehyde and its sulfobutyl ether-ß-cyclodextrin inclusion complex and their application in fish preservation.

For food preservation, the packaging film needs to have higher antibacterial activity in initial phase and keep longer activity. In this study, cinnamaldehyde (CA) and its sulfobutyl ether-ß-cyclodextrin inclusion complex (CA/S) were used to fabricate fish gelatin antibacterial composite films. The addition enhanced the elongation at break and light barrier property of the films. Film forming solution incorporated with CA and CA/S presented the most excellent inhibition ratio against Pseudomonas aeruginosa, which was 98.43 ± 1.11% in initial period and still 82.97 ± 4.55% at 72 h. Further, the packaging solution of gelatin combined CA and CA/S effectively inhibited the growth of microorganisms during preservation of grass carp slices. Especially, the total volatile salt-based nitrogen (TVB-N) did not exceed 10 mg/100 g at the end of storage, indicating that the active coating could obviously extend the shelf life of fish muscle. This work provided a promising food packaging system with antimicrobial and environmentally friendly.

Crystal Structure of the Extracellular Domains of GPR110.

Adhesion G protein-coupled receptors (aGPCRs) play a pivotal role in human immune responses, cellular communication, organ development, and other processes. GPR110 belongs to the aGPCR subfamily VI and was initially identified as an oncogene involved in lung and prostate cancers. GPR110 contains tandem adhesion domains at the extracellular region that mediate inter-cellular signaling. However, the structural organization and signaling mechanism for these tandem domains remain unclear. Here, we report the crystal structure of a GPR110 fragment composing the SEA, HormR, and GAIN domains at 2.9 Å resolution. The structure together with MD simulations reveal rigid connections between these domains that are stabilized by complementary interfaces. Strikingly, we found N-linked carbohydrates attached to N389 of the GAIN domain form extensive contacts with the preceding HormR domain. These interactions appear to be critical for folding, as removal of the glycosylation site greatly decreases expression of the GPR110 extracellular fragment. We further demonstrate that the ligand synaptamide fits well within the hydrophobic pocket occupied by the Stachel peptide in the rest state. This suggests that the agonist may function by removing the Stachel peptide which in turn redocks to the orthosteric pocket for receptor activation. Taken together, our structural findings and analyses provide novel insights into the activation mechanism for aGPCRs.

Engineering a precise adenine base editor with minimal bystander editing.

Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise safety concerns. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1-2 nucleotides with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited the A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision over ABE8e) and was further confirmed through a library of guide RNA-target sequence pairs. Owing to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic single-nucleotide variants when fused to Cas9 variants with expanded protospacer adjacent motif compatibility. bpNLS, bipartite nuclear localization signals.

Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing.

Cytosine base editors (CBEs) efficiently generate precise C·G-to-T·A base conversions, but the activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family deaminase component induces considerable off-target effects and indels. To explore unnatural cytosine deaminases, we repurpose the adenine deaminase TadA-8e for cytosine conversion. The introduction of an N46L variant in TadA-8e eliminates its adenine deaminase activity and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of highly efficient and precise C·G-to-G·C editing. Through fusion with uracil glycosylase inhibitors and further introduction of additional variants, a series of Td-CBEs was obtained either with a high activity similar to that of BE4max or with higher precision compared to other reported accurate CBEs. Td-CGBE/Td-CBEs show very low indel effects and a background level of Cas9-dependent or Cas9-independent DNA/RNA off-target editing. Moreover, Td-CGBE/Td-CBEs are more efficient in generating accurate edits in homopolymeric cytosine sites in cells or mouse embryos, suggesting their accuracy and safety for gene therapy and other applications.

Cryo-EM structure of the human adenosine A2B receptor-Gs signaling complex.

The human adenosine A2B receptor (A2BR) is a class A G protein-coupled receptor that is involved in several major physiological and pathological processes throughout the body. A2BR recognizes its ligands adenosine and NECA with relatively low affinity, but the detailed mechanism for its ligand recognition and signaling is still elusive. Here, we present two structures determined by cryo-electron microscopy of A2BR bound to its agonists NECA and BAY60-6583, each coupled to an engineered Gs protein. The structures reveal conserved orthosteric binding pockets with subtle differences, whereas the selectivity or specificity can mainly be attributed to regions extended from the orthosteric pocket. We also found that BAY60-6583 occupies a secondary pocket, where residues V2506.51 and N2737.36 were two key determinants for its selectivity against A2BR. This study offers a better understanding of ligand selectivity for the adenosine receptor family and provides a structural template for further development of A2BR ligands for related diseases.

Apoptin inhibits glycolysis and regulates autophagy by targeting pyruvate kinase M2 (PKM2) in lung cancer A549 cells.

Pyruvate kinase M2 (PKM2) is a key enzyme in aerobic glycolysis, and which plays an important role in tumor energy metabolism and tumor growth. Ad-apoptin, a recombinant oncolytic adenovirus, that can stably express apoptin in tumor cells and selectively causes cell death in tumor cells. The relationship between the anti-tumor function of apoptin, including apoptosis and autophagy activation, and energy metabolism of tumor cells has not been clarified. In this study, we used the A549 lung cancer cell line to analyze the mechanism of PKM2 involvement apoptin-mediated cell death in tumor cells. PKM2 expression in lung cancer cells was detected by Western blot and qRT-PCR. In the PKM2 knockdown and over-expression experiments, A549 lung cancer cells were treated with Ad-apoptin, and cell viability was determined by the CCK-8 assay and crystal violet staining. Glycolysis was investigated using glucose consumption and lactate production experiments. Moreover, the effects of Ad-apoptin on autophagy and apoptosis were analyzed by immunofluorescence using the Annexin v-mCherry staining and by western blot for c-PARP, p62 and LC3-II proteins. Immunoprecipitation analysis was used to investigate the interaction between apoptin and PKM2. In addition, following PKM2 knockdown and overexpression, the expression levels of p-AMPK, p-mTOR, p-ULK1, and p-4E-BP1 proteins in Ad-apoptin treated tumor cells, were analyzed by western blot to investigate the mechanism of apoptin effect on the energy metabolism of tumor cells. The in vivo antitumor mechanism of apoptin was analyzed by xenograft tumor inhibition experiment in nude mice and immunohistochemistry of tumors' tissue. As a result, apoptin could target PKM2, inhibit glycolysis and cell proliferation in A549 cells, and promote autophagy and apoptosis in A549 cells by regulating the PKM2/AMPK/mTOR pathway. This study confirmed the necessary role of Ad-apoptin in energy metabolism of A549 cells.

Apoptin mediates mitophagy and endogenous apoptosis by regulating the level of ROS in hepatocellular carcinoma.

BACKGROUND: Apoptin, as a tumor-specific pro-apoptotic protein, plays an important anti-tumoral role, but its mechanism of autophagy activation and the interaction between autophagy and apoptosis have not been accurately elucidated. Here, we studied the mechanism of apoptin-induced apoptosis and autophagy and the interaction between two processes. METHODS: Using crystal violet staining and the CCK-8 assay, we analyzed the effect of apoptin in the inhibition of liver cancer cells in vitro and analyzed the effect of inhibiting liver cancer in vivo by establishing a nude mouse tumor model. Flow cytometry and fluorescence staining were used to analyze the main types of apoptin-induced apoptosis and autophagy. Subsequently, the relationship between the two events was also analyzed. Flow cytometry was used to analyze the effect of ROS on apoptin-mediated apoptosis and autophagy mediated by apoptin. The effect of ROS on two phenomena was analyzed. Finally, the role of key genes involved in autophagy was analyzed using gene silencing. RESULTS: The results showed that apoptin can significantly increase the apoptosis and autophagy of liver cancer cells, and that apoptin can cause mitophagy through the increase in the expression of NIX protein. Apoptin can also significantly increase the level of cellular ROS, involved in apoptin-mediated autophagy and apoptosis of liver cancer cells. The change of ROS may be a key factor causing apoptosis and autophagy. CONCLUSION: The above results indicate that the increase in ROS levels after apoptin treatment of liver cancer cells leads to the loss of mitochondrial transmembrane potential, resulting in endogenous apoptosis and mitophagy through the recruitment of NIX. Therefore, ROS may be a key factor connecting endogenous apoptosis and autophagy induced by apoptin in liver cancer cells. Video abstract.

Caryophyllene Oxide Induces Ferritinophagy by Regulating the NCOA4/FTH1/LC3 Pathway in Hepatocellular Carcinoma.

Ferritinophagy is associated with tumor occurrence, development, and therapy effects. Ferritinophagy and ferroptosis are regulated by iron metabolism and are closely connected. LC3 protein is a key protein in autophagy. Following the binding of NCOA4 to FTH1, it links to LC3Ⅱ in lysosomes, a symbol of ferritinophagy. A ferritinophagy's inducer is likely to open new avenues for anticancer medication research and development. In this study, we discovered that caryophyllene oxide has a substantial inhibitory effect on HCCLM3 and HUH7 cells, by regulating the level of cellular oxidative stress, and the levels of autophagy and iron metabolism in HCCLM3 and HUH7 cells, leading to a ferritinophagy-related phenomenon. Furthermore, the results of T-AOC, DPPH free radical scavenging rate, and hydroxyl radical inhibition indicated that caryophyllene oxide can inhibit cell anti-oxidation. The examination of the ferritinophagy-related process revealed that caryophyllene oxide promotes the production and accumulation of intracellular reactive oxygen species and lipid peroxidation. NCOA4, FTH1, and LC3Ⅱ were found to be targeted regulators of caryophyllene oxide. Caryophyllene oxide regulated NCOA4, LC3 Ⅱ, and FTH1 to promote ferritinophagy. In vivo, we discovered that caryophyllene oxide can lower tumor volume, significantly improve NCOA4 and LC3 protein levels in tumor tissue, and raise Fe2+ and malondialdehyde levels in serum. The compound can also reduce NRF2, GPX4, HO-1, and FTH1 expression levels. The reduction in the expression levels of NRF2, GPX4, HO-1, and FTH1 by caryophyllene oxide also inhibited GSH and hydroxyl radical's inhibitory capacities in serum, and promoted iron deposition in tumor tissue resulting in the inhibition of tumor growth. In summary, our study revealed that caryophyllene oxide mostly kills liver cancer cells through ferritinophagy-mediated ferroptosis mechanisms. In conclusion, caryophyllene oxide may be used as a ferritinophagy activator in the field of antitumor drug research and development.

Crystal structure of a constitutive active mutant of adenosine A2A receptor.

The adenosine A2A receptor (A2AAR) is a prototypical member of the class A subfamily of G-protein-coupled receptors (GPCRs) that is widely distributed in various tissues and organs of the human body, and participates in many important signal-regulation processes. We have previously summarized a common activation pathway of class A GPCRs in which a series of conserved residues/motifs undergo conformational change during extracellular agonist binding and finally induce the coupling of intracellular G protein. Through this mechanism we have successfully predicted several novel constitutive active or inactive mutations for A2AAR. To reveal the molecular mechanism of mutation-induced constitutive activity, we determined the structure of a typical mutant I92N complexed with the agonist UK-432097. The mutated I92N forms a hydrophilic interaction network with nearby residues including Trp6.48 of the CWxP motif, which is absent in wild-type A2AAR. Although the mutant structure is similar overall to the previously determined intermediate-state A2AAR structure (PDB ID 3qak) [Xu, Wu, Katritch, Han, Jacobson, Gao, Cherezov & Stevens (2011). Science, 332, 322-327 ▸], molecular dynamics simulations suggest that the I92N mutant stabilizes the metastable intermediate state through the hydrophilic interaction network and favors the conformational transition of the receptor towards the active state. This research provides a structural template towards the special pharmacological outcome triggered by conformational mutation and sheds light on future structural or pharmaco-logical studies among class A GPCRs.

Ad-VT enhances the sensitivity of chemotherapy-resistant lung adenocarcinoma cells to gemcitabine and paclitaxel in vitro and in vivo.

Background One of the main challenges in the clinical treatment of lung cancer is resistance to chemotherapeutic drugs. P-glycoprotein (P-gp)-mediated drug resistance is the main obstacle to successfully implementing microtubule-targeted tumor chemotherapy. Purpose In this study, we explored the effect of Ad-hTERTp-E1a-Apoptin (Ad-VT) on drug-resistant cell lines and the molecular mechanism by which Ad-VT combined with chemotherapy affects drug-resistant cells and parental cells. Methods In vitro, cell proliferation, colony formation, resistance index (RI), apoptosis and autophagy assays were performed. Protein expression was analyzed by Western blotting. Finally, a xenograft tumor model in nude mice was used to detect tumor growth and evaluate histological characteristics. Results Our results showed that Ad-VT had an obvious killing effect on A549, A549/GEM and A549/Paclitaxel cancer cells, and the sensitivity of drug-resistant cell lines to Ad-VT was significantly higher than that of parental A549 cells. Compared with A549 cells, A549/GEM and A549/Paclitaxel cells had higher autophagy levels and higher viral replication ability. Ad-VT decreased the levels of p-PI3k, p-Akt and p-mTOR and the expression of P-gp. In vivo, Ad-VT combined with chemotherapy can effectively inhibit the growth of chemotherapy-resistant tumors and prolong the survival of mice. Conclusions Thus, the combination of Ad-VT and chemotherapeutic drugs will be a promising strategy to overcome chemoresistance.