Circular RNA-DPP4 serves an oncogenic role in prostate cancer progression through regulating miR-195/cyclin D1 axis.

BACKGROUND: Recently, more and more studies have highlighted the critical regulatory roles of circular RNAs (circRNAs), a class of non-coding RNAs, in the progression of many human cancers, including prostate cancer (PCa). circRNA microarray analysis was performed to identify circRNAs that are differentially expressed in PCa tissues. METHODS: 104 pairs of PCa tissues and matched adjacent normal prostate tissues (at least 2 cm distal to the tumor margin) were obtained. circRNA microarray analysis was performed on four pairs of PCa tissues and matched adjacent normal prostate tissues to investigate the potential involvement of circRNAs in PCa. Flow cytometric analysis was performed to investigate whether the effect of circDPP4 on PCa cell proliferation was associated with the alteration in cell cycle progression. The role of circDPP4 in PCa tumor growth was further explored in vivo. RESULTS: We found that circDPP4 was overexpressed in PCa tissues and cell lines, and its expression was closely associated with Gleason score and clinical stage of PCa patients. In vitro loss- and gain-of-function experiments demonstrated that circDPP4 knockdown inhibited, whereas circDPP4 overexpression promoted the proliferation, migration, invasion and cell cycle progression of PCa cells. Knockdown of circDPP4 also suppressed PCa tumor growth in vivo. We further found that circDPP4 functioned as a competing endogenous RNA (ceRNA) for miR-195 in PCa cells, and miR-195 negatively regulated the expression of oncogenic cyclin D1. Rescue experiments suggested that restoration of miR-195 blocked the oncogenic role of circDPP4 in PCa cells. CONCLUSIONS: Taken together, our findings revealed a novel regulatory mechanism between circDPP4 and miR-195/cyclin D1 axis, and offered novel strategies for the treatment of PCa.

Genome-wide mapping of 5-hydroxymethylcytosines in circulating cell-free DNA as a non-invasive approach for early detection of hepatocellular carcinoma.

OBJECTIVE: The lack of highly sensitive and specific diagnostic biomarkers is a major contributor to the poor outcomes of patients with hepatocellular carcinoma (HCC). We sought to develop a non-invasive diagnostic approach using circulating cell-free DNA (cfDNA) for the early detection of HCC. DESIGN: Applying the 5hmC-Seal technique, we obtained genome-wide 5-hydroxymethylcytosines (5hmC) in cfDNA samples from 2554 Chinese subjects: 1204 patients with HCC, 392 patients with chronic hepatitis B virus infection (CHB) or liver cirrhosis (LC) and 958 healthy individuals and patients with benign liver lesions. A diagnostic model for early HCC was developed through case-control analyses using the elastic net regularisation for feature selection. RESULTS: The 5hmC-Seal data from patients with HCC showed a genome-wide distribution enriched with liver-derived enhancer marks. We developed a 32-gene diagnostic model that accurately distinguished early HCC (stage 0/A) based on the Barcelona Clinic Liver Cancer staging system from non-HCC (validation set: area under curve (AUC)=88.4%; (95% CI 85.8% to 91.1%)), showing superior performance over α-fetoprotein (AFP). Besides detecting patients with early stage or small tumours (eg, ≤2.0 cm) from non-HCC, the 5hmC model showed high capacity for distinguishing early HCC from high risk subjects with CHB or LC history (validation set: AUC=84.6%; (95% CI 80.6% to 88.7%)), also significantly outperforming AFP. Furthermore, the 5hmC diagnostic model appeared to be independent from potential confounders (eg, smoking/alcohol intake history). CONCLUSION: We have developed and validated a non-invasive approach with clinical application potential for the early detection of HCC that are still surgically resectable in high risk individuals.

Droplet-digital polymerase chain reaction for detection of clinical hepatitis B virus DNA samples.

This study aimed to establish a quantitative droplet-digital polymerase chain reaction (ddPCR) detection system for clinical hepatitis B virus (HBV) DNA in samples from patients with HBV infection with the aim of monitoring fluctuations and planning future antiviral treatments. Primers and probes for the conserved region of HBV DNA sequence (GenBank: AB554017.1) were designed by sequence alignment with the HBV DNA sequence in GenBank. The PUC57 plasmid containing the gene fragment was then synthesized according to the fragment amplified by the primer. Linear DNA templates were obtained by digestion of the PUC57 plasmid with EcoRI. Primer specificity was verified by electrophoresis and sequencing, and the plasmid was used to verify the performance of ddPCR. Serum levels of HBV DNA were then detected in 103 HBV patients by ddPCR and real-time quantitative polymerase chain reaction (qPCR), and the consistency of the two methods was compared. The accuracy of the ddPCR assays showed good linear correlation (R2 = 0.9952). The ddPCR coefficient of variance (CV) for intrarun replicates reached 2.0%, and the CV value for inter-run replicates reached 3.6%. The ddPCR could detect as little as one copy of the template, as demonstrated by continuous dilution. This assay system provided a novel and specific platform with a linear range of detection from 1 to 105 copies/µL. Serum samples were collected from 103 HBV patients, and HBV DNA levels quantified by ddPCR and qPCR showed good concordance based on the Bland-Altman analysis. The 95% limits of agreement were log10 : 0.29 ± 0.55 copies/µL. Our findings demonstrated that ddPCR can improve the detection level of nucleic acid and quantify the nucleic acid concentration, which is suitable for the clinical detection of HBV DNA.

17ß-estradiol (E2) in membranes: Orientation and dynamic properties.

Non-genomic membrane effects of estrogens are of great interest because of the diverse biological activities they may elicit. To further our understanding of the molecular features of the interaction between estrogenic hormones and membrane bilayers, we have determined the preferred orientation, location, and dynamic properties of 17ß-estradiol (E2) in two different phospholipid membrane environments using (2)H-NMR and 2D (1)H-(13)C HSQC in conjunction with molecular dynamics simulations. Unequivocal spectral assignments to specific (2)H labels were made possible by synthesizing six selectively deuterated E2 molecules. The data allow us to conclude that the E2 molecule adopts a nearly "horizontal" orientation in the membrane bilayer with its long axis essentially perpendicular to the lipid acyl-chains. All four rings of the E2 molecule are located near the membrane interface, allowing both the E2 3-OH and the 17ß-OH groups to engage in hydrogen bonding and electrostatic interactions with polar phospholipid groups. The findings augment our knowledge of the molecular interactions between E2 and membrane bilayer and highlight the asymmetric nature of the dynamic motions of the rigid E2 molecule in a membrane environment.

The interaction of cannabinoid receptor agonists, CP55940 and WIN55212-2 with membranes using solid state 2H NMR.

Two key commonly used cannabinergic agonists, CP55940 and WIN55212-2, are investigated for their effects on the lipid membrane bilayer using (2)H solid state NMR, and the results are compared with our earlier work with delta-9-tetrahydrocannabinol (Δ(9)-THC). To study the effects of these ligands we used hydrated bilayers of dipalmitoylphosphatidylcholine (DPPC) deuterated at the 2' and 16' positions of both acyl chains with deuterium atoms serving as probes for the dynamic and phase changes at the membrane interface and at the bilayer center respectively. All three cannabinergic ligands lower the phospholipid membrane phase transition temperature, increase the lipid sn-2 chain order parameter at the membrane interface and decrease the order at the center of the bilayer. Our studies show that the cannabinoid ligands induce lateral phase separation in the lipid membrane at physiological temperatures. During the lipid membrane phase transition, the cooperative dynamic process whereby the C-(2)H segments at the interface and center of the bilayer spontaneously reach the fast exchange regime ((2)H NMR timescale) is distinctively modulated by the two cannabinoids. Specifically, CP55940 is slightly more efficient at inducing liquid crystalline-type (2)H NMR spectral features at the membrane interface compared to WIN55212-2. In contrast, WIN55212-2 has a far superior ability to induce liquid crystalline-type spectral features at the center of the bilayer, and it increases the order parameter of the sn-1 chain in addition to the sn-2 chain of the lipids. These observations suggest the cannabinoid ligands may influence lipid membrane domain formations and there may be contributions to their cannabinergic activities through lipid membrane microdomain related mechanisms. Our work demonstrates that experimental design strategies utilizing specifically deuterium labeled lipids yield more detailed insights concerning the properties of lipid bilayers.

Screening of MicroRNA Biomarkers Related to the Progression of Bladder Cancer.

Objective: This study aimed to identify the novel microRNAs (miRNAs) for early diagnosis of bladder cancer. Materials and Methods: Differentially expressed miRNAs between early and advanced bladder cancer were identified by differential expression analysis, using miRNA-seq data from The Cancer Genome Atlas (TCGA). The optimal subset of feature miRNAs for pathologic stage prediction was acquired by Random Forest algorithm and was used to construct a support vector machine (SVM) classifier. The performance of the SVM classifier in predicting the progression of bladder cancer samples was validated using an independent validating dataset. An miRNA-regulated target gene network was finally constructed and functional annotation were performed for the target genes. Results: A total of 52 significantly differentially expressed miRNAs were identified between early and advanced bladder cancer samples and 17 of these miRNAs were identified to be feature miRNAs. The 17 feature miRNAs were used to construct an SVM classifier, which showed a high performance in pathologic stage prediction for both training and validating dataset. Besides, our functional annotation analysis showed that the feature miRNAs were significantly involved in biological processes and pathways related to extracellular matrix process and PI3K/Akt signaling. Conclusions: The optimal subset of miRNAs may act as potential therapeutic targets and diagnostic markers of bladder cancer.

The lncRNA OGFRP1/miR-149-5p/IL-6 axis regulates prostate cancer chemoresistance.

BACKGROUND: The long non-coding RNA (lncRNA) OGFRP1 has been found to promote malignancy in prostate cancer (PC) and other cancer types. How this lncRNA functions in the regulation of PC chemoresistance, however, is poorly defined. METHODS: qRT-PCR was employed to measure OGFRP1, miR-149-5p, and IL-6 expression in PC tissues and cells. IC50 values for paclitaxel and docetaxel in PC cells were assessed via a CCK-8 assay approach. Putative miR-149-5p binding targets were identified and validated through bioinformatics assays and luciferase reporter assays, respectively. The impact of OGFRP1 on PC chemoresistance in vivo was validated using a xenograft model system. RESULTS: Docetaxel-resistant PC (PC/DR) cells and tissues exhibited reduced OGFRP1 expression and increased miR-149-5p expression. Knocking down OGFRP1 augmented the sensitivity of these PC cells to docetaxel and paclitaxel in vitro and in vivo. Mechanistically, OGFRP1 was found to bind and sequester miR-149-5p within PC/DR cells, thereby indirectly regulating IL-6 expression. Consistent with this model, the overexpression of IL-6 reversed the OGFRP1 knockdown-mediated reductions in docetaxel and paclitaxel IC50 values for these PC cells. CONCLUSIONS: OGFRP1 can sequester miR-149-5p, thereby indirectly promoting IL-6 upregulation and thereby promoting chemoresistance in PC cells. This OGFRP1/miR-149-5p/IL-6 axis may thus be a promising target for therapeutic efforts aimed at PC chemosensitization and treatment.

Efficacy and safety of Hou Gu Mi Xi for peptic ulcer diseases: Study protocol for a randomized controlled trial.

BACKGROUND: Peptic ulcer disease (PUD) is a major burden worldwide. Several challenges remain with standard Western treatment of PUD, such as persistent weakness, fatigue, and relapse. A dietary traditional Chinese medicine (TCM) formula, Hou Gu Mi Xi (HGMX), has been developed as a complementary treatment for PUD. AIMS: This multicenter, double-blind, randomized controlled trial will assess efficacy and safety of HGMX in patients with PUD. METHODS: Three hundred sixty eligible patients will be assigned to receive HGMX, placebo, HGMX + rabeprazole or placebo + rabeprazole for 4 weeks after 2 weeks of standard Western treatment. This first step, with a 2 × 2 factorial design, will focus on assessing the main and interaction effects of HGMX and rabeprazole on ulcer healing. Then, rabeprazole will be stopped, and HGMX will be continued for up to 1 year. The second step, with a placebo-controlled design, will compare the long-term effects of HGMX and placebo. Extended follow-up with no treatment will continue for up to 2 years. Independent and paired t tests, Pearson χ test and the rank-sum test will be used to compare between-group differences. The P value will be adjusted using the O'Brien & Fleming method for multiple comparisons. EXPECTED OUTCOMES: The primary outcomes are total efficacy rate of PUD treatment, quality of ulcer healing, and changes in spleen qi deficiency symptoms. The secondary outcomes include ulcer area, PUD recurrence, Helicobacter pylori eradication rate, gastric function, body weight, and body mass index. Adverse events (AEs), severe AEs, treatment-related AEs, and withdrawal owing to AEs will be recorded to assess treatment safety. DISCUSSION: The trial results will provide high-quality evidence for HGMX, as a complementary therapy, for the long-term management of PUD and will be valuable for the development of related guidelines and regulations. TRIAL REGISTRATION: The protocol of this trial was approved in all research hospitals and was registered in ClinicalTrials.gov at October 25, 2017(No. NCT03320538).

Efficacy and Safety of Hou Gu Mi Xi on Spleen Qi Deficiency in Patients with Nonorganic Gastrointestinal Disorders: Protocol for a Multicenter, Randomized, Placebo-Controlled Trial.

BACKGROUND: There is a worldwide epidemic of nonorganic gastrointestinal disorders (NOGDs), which are a class of disorders that cause various discomforts and ultimately progress into organic gastrointestinal diseases. Because of the unsatisfactory efficacy of Western medical treatments, traditional Chinese medicine (TCM) is becoming a promising complementary and alternative treatment to manage NOGDs. OBJECTIVES: To investigate the efficacy and safety of Hou Gu Mi Xi (HGMX), a newly developed dietary TCM formula, on the syndrome of spleen qi deficiency (SQD) in patients with NOGDs. METHODS/DESIGN: This study is a multicenter, randomized, double-blinded, parallel, and placebo-controlled trial that will last for 2 years. All qualified subjects with NOGDs and SQD will be included. The study population will be divided into the HGMX and placebo groups. To assess the efficacy of HGMX, we will mainly focus on changes in SQD symptoms scored by a Spleen Qi Deficiency Symptoms Grading and Quantifying Scale and evaluate changes in gastrin-17, the negative Helicobacter pylori conversion rate, body weight, body mass index, and gastroscopy findings. The safety of HGMX will be assessed by recording adverse events (AEs), severe AEs, treatment-related AEs and withdrawal due to AEs. DISCUSSION: This trial is part of our study series that intends to validate the potential of HGMX in the management of chronic gastrointestinal diseases. This series of RCTs is the first committed to the evaluation of a dietary TCM formula and will hopefully establish an evidence-based clinical research model for dietary TCM formulas. ETHICS: The protocol was approved by Ethics Committee of five research hospitals and was registered in Clinicaltrials.gov (NCT03019042).

[A study of environmental changes on new snail habitat though multi-temporal relative radiometric normalized TM images].

OBJECTIVE: To study the environmental changes of new snail habitual by using multi-temporal relative radiometric normalized TM images taken in Wuzhoutou of Nanchang, Jiangxi province. METHODS: TM images of Poyang Lake were collected on 3rd April 1998, 16th April 2000 and 5th May 2004. Taking images in 2004 as a reference, the TM images in 1998 and 2000 were radiometric normalized. The images were analyzed to trace the vegetation background and snail historical information. RESULTS: The vegetation coverage rate of Wuzhaoutou on April 3rd in 1998 was 4.76%, but the vegetation coverage rates of Wuzhoutou on April 16th in 2000 and May 5th in 2004 were above 80%; the NDVI values of Wuzhoutou on April 3rd in 1998 were around -0.4 to 0.2, but the NDVI values of Wuzhoutou on April 16th in 2000 and May 5th in 2004 were mainly around 0.2 to 0.6, which had statistical difference with that in 1998 (q = 162.285321, P < 0.01 and q = 161.669725, P < 0.01). The greenness of three images have statistical differences(q = 178.679245, P < 0.01; q = 130.320755, P < 0.01 and q = - 48.358491, P < 0.01). CONCLUSION: Combining with the snail survey, the TM images could be an effective measure for setting up models to forecast the areas where snails might live to control the Schistosomiasis epidemic.

[Application of global positioning system and geographic information system in surveillance of schistosomiasis].

OBJECTIVE: To use the snail survey data of Yugan, Jiangxi Province as an example to evaluate the effects and advantages of global positioning system integrated with digital maps and remote sensing data in order to manage and analyze the whole country's surveillance data of schistosomiasis by using geographic information system. METHODS: The data of geographic information and snail information of 20 marshlands in Yugan, Jiangxi Province were collected, and by the correction of remote sensing images based on the digital maps and index extraction, the snail current situation and trend were analyzed and expressed. RESULTS: A series of specific procedure images of these 20 marshlands was established and the effects of Yugan surveillance platform based on geographic information system were validated. CONCLUSION: The whole country's surveillance data of schistosomiasis might be successfully integrated with geographic information system by global positioning system and be managed and expressed, which supplies a successful platform in using the surveillance data effectively.

Hydrogen-bonded His93 as a sensitive probe for identifying inhibitors of the endocannabinoid transport protein FABP7.

The human brain FABP (FABP7) has been shown to be an intracellular carrier protein that can significantly potentiate the uptake of the endocannabinoid anandamide. For this reason, there is a great interest in the discovery and development of FABP7 inhibitors for treating stress, pain, inflammation, and drug abuse. We found that in the (1) H-NMR spectrum of the protein, a well-separated downfield resonance arising from the hydrogen-bonded His93 side chain is very sensitive to ligand binding. Using this characteristic spectral marker together with another well-resolved upfield resonance from the side chain of Val84, we have identified that an adipocyte FABP (FABP4) inhibitor BMS309403 also binds tightly to FABP7. Our data demonstrated that this unique His93 downfield resonance can be used as a sensitive probe for rapidly and unambiguously identifying novel high-affinity FABP7 ligands. The findings should help accelerate the discovery of potential drug leads for the modulation of endocannabinoid transport.

Conformational study of lipophilic ligands in phospholipid model membrane systems by solution NMR.

Phospholipid bicelles were employed as a membrane bilayer model in the conformational studies of two lipophilic cannabinoids, delta(8)-THC and its O-methyl ether analogue, Me-Delta(8)-THC using conventional high-resolution NMR. A preparation of 8% (w/v) phospholipid concentration and a high DMPC/DHPC ratio (q = 2.0) was found to be optimal for not only effectively incorporating our ligands, but also providing a more bilayerlike environment suitable for conformational studies. While the conformational differences between the two cannabinoids could not be observed in chloroform and were barely detectable in SDS micelle solution, there is an increasing preference for the pentyl tail of Delta(8)-THC to bend toward the tricyclic ring system with increasing proportions of DMPC in the bicelle preparation. Our results highlight the advantages of exploring the conformational properties of cannabinoids using bicelle preparations as a medium that more closely resembles biological membrane bilayers and eliminates the need for isotopic labeling. This approach should also be of more general value for studying the interactions of other cannabinoids and biologically active, hydrophobic or amphipathic, small molecules with membranes.

The interaction of fatty acid amide hydrolase (FAAH) inhibitors with an anandamide carrier protein using (19)F-NMR.

It has been reported that the endocannabinoid anandamide (AEA) binds to a class of fatty acid-binding proteins and serum albumin which can serve as carrier proteins and potentiate the cellular uptake of AEA and its intracellular translocation. Here, we employed (19)F nuclear magnetic resonance spectroscopy to study the interactions of serum albumin with two inhibitors of fatty acid amide hydrolase (FAAH), the enzyme involved in the deactivation of anandamide. We found that, for both inhibitors AM5206 and AM5207, the primary binding site on serum albumin is drug site 1 located at subdomain IIA. Neither inhibitor binds to drug site 2. While AM5207 binds exclusively to drug site 1, AM5206 also interacts with other fatty acid-binding sites on serum albumin. Additionally, AM5206 has an affinity for serum albumin approximately one order of magnitude higher than that of AM5207. The data suggest that interactions of FAAH inhibitors with albumin may provide added advantages for their ability to modulate endocannabinoid levels for a range of applications including analgesia, antiemesis, and neuroprotection.

Targeting the Endocannabinoid System for Neuroprotection: A 19F-NMR Study of a Selective FAAH Inhibitor Binding with an Anandamide Carrier Protein, HSA.

Fatty acid amide hydrolase (FAAH), the enzyme involved in the inactivation of the endocannabinoid anandamide (AEA), is being considered as a therapeutic target for analgesia and neuroprotection. We have developed a brain permeable FAAH inhibitor, AM5206, which has served as a valuable pharmacological tool to explore neuroprotective effects of this class of compounds. In the present work, we characterized the interactions of AM5206 with a representative AEA carrier protein, human serum albumin (HSA), using 19F nuclear magnetic resonance (NMR) spectroscopy. Our data showed that as a drug carrier, albumin can significantly enhance the solubility of AM5206 in aqueous environment. Through a series of titration and competitive binding experiments, we also identified that AM5206 primarily binds to two distinct sites within HSA. Our results may provide insight into the mechanism of HSA-AM5206 interactions. The findings should also help in the development of suitable formulations of the lipophilic AM5206 and its congeners for their effective delivery to specific target sites in the brain.

[Gastroscopic and pathological characteristics of patients with or without schistosomiasis in Poyang Lake area].

OBJECTIVES: To understand the gastroscopic and pathological characteristics of patients with miasis in Poyang Lake area, and to explore the relationship between schistosomiasis and pathological changes of gastric mucosa. METHODS: Volunteers with or without schistosomiasis were recruited and divided into a case group and a control group. All the objects were examined by electronic gastroscopy and pathological examinations. RESULTS: Two hundred and fifty-three volunteers diagnosed with chronic or advanced schistosomiasis in the case group showed different degrees of gastric mucosal changes, including 7 cases with schistosomal eggs deposited beneath the gastric mucosa (with an incidence of 2.77%) , 33 cases with dysplastic hyperplasia and intestinal metaplasia (with an incidence of precancerous lesion of 13.04%), and 1 case with gastric cancer. While in the 200 volunteers without schistosomiasis in the control group, the results showed milder gastric mucosal changes, 33 cases were detected with dysplastic hyperplasia and intestinal metaplasia (with an incidence of 7.50%), and 1 case was diagnosed gastric cancer. The difference between the incidences of precancerous lesion in the two groups had no statistic significance (P > 0.05). CONCLUSIONS: The incidence and extent of gastric mucosal changes in schistosomiasis patients are higher and more serious than those in non-schistosomiasis patients, and gastrointestinal schistosomiasis is not related to gastric cancer.

Magnetically aligned bicelles to study the orientation of lipophilic ligands in membrane bilayers.

Magnetically aligned bicelles were used as a model membrane to study the orientation and dynamic properties of two cannabinoids (Delta (8)-THC and Me-Delta (8)-THC) using (31)P and (2)H NMR. The uniform alignment of the bicelles allowed us to obtain well resolved deuterium spectra from a solution NMR spectrometer. The preferred orientations of Delta (8)-THC and Me-Delta (8)-THC were calculated on the basis of the measurements of individual quadrupolar splittings. Our results agree with previous experiments using multilamellar membranes as well as with molecular dynamics simulation data described here. In conjunction with our earlier report using small and fast tumbling bicelles, the present work of well aligned bicelles shows that bicelle preparations can provide either pseudoisotropic or anisotropic NMR spectra to study the conformation, orientation, and dynamic properties of ligands in membrane bilayers. Such data are of critical value for understanding the interactions of lipophilic drug molecules with membrane proteins.

The conformation, location, and dynamic properties of the endocannabinoid ligand anandamide in a membrane bilayer.

The endogenous cannabinoid ligand anandamide is biosynthesized from membrane phospholipid precursors and is believed to reach its sites of action on the CB1 and CB2 receptors through fast lateral diffusion within the cell membrane. To gain a better insight on the stereochemical features of its association with the cell membrane and its interaction with the cannabinoid receptors, we have studied its conformation, location, and dynamic properties in a dipalmitoylphosphatidylcholine multilamellar model membrane bilayer system. By exploiting the bilayer lattice as an internal three-dimensional reference grid, the conformation and location of anandamide were determined by measuring selected inter- and intramolecular distances between strategically introduced isotopic labels using the rotational echo double resonance (REDOR) NMR method. A molecular model was proposed to represent the structural features of our anandamide/lipid system and was subsequently used in calculating the multispin dephasing curves. Our results demonstrate that anandamide adopts an extended conformation within the membrane with its headgroup at the level of the phospholipid polar group and its terminal methyl group near the bilayer center. Parallel static (2)H NMR experiments further confirmed these findings and provided evidence that anandamide experiences dynamic properties similar to those of the membrane phospholipids and produces no perturbation to the bilayer. Our results are congruent with a hypothesis that anandamide approaches its binding site by laterally diffusing within one membrane leaflet in an extended conformation and interacts with a hydrophobic groove formed by helices 3 and 6 of CB1, where its terminal carbon is positioned close to a key cysteine residue in helix 6 leading to receptor activation.