Structure-based identification of a G protein-biased allosteric modulator of cannabinoid receptor CB1.

Cannabis sativa is known for its therapeutic benefit in various diseases including pain relief by targeting cannabinoid receptors. The primary component of cannabis, Δ9-tetrahydrocannabinol (THC), and other agonists engage the orthosteric site of CB1, activating both Gi and ß-arrestin signaling pathways. The activation of diverse pathways could result in on-target side effects and cannabis addiction, which may hinder therapeutic potential. A significant challenge in pharmacology is the design of a ligand that can modulate specific signaling of CB1. By leveraging insights from the structure-function selectivity relationship (SFSR), we have identified Gi signaling-biased agonist-allosteric modulators (ago-BAMs). Further, two cryoelectron microscopy (cryo-EM) structures reveal the binding mode of ago-BAM at the extrahelical allosteric site of CB1. Combining mutagenesis and pharmacological studies, we elucidated the detailed mechanism of ago-BAM-mediated biased signaling. Notably, ago-BAM CB-05 demonstrated analgesic efficacy with fewer side effects, minimal drug toxicity and no cannabis addiction in mouse pain models. In summary, our finding not only suggests that ago-BAMs of CB1 provide a potential nonopioid strategy for pain management but also sheds light on BAM identification for GPCRs.

Cryo-EM structures of human GPR34 enable the identification of selective antagonists.

GPR34 is a functional G-protein-coupled receptor of Lysophosphatidylserine (LysoPS), and has pathogenic roles in numerous diseases, yet remains poorly targeted. We herein report a cryo-electron microscopy (cryo-EM) structure of GPR34 bound with LysoPS (18:1) and Gi protein, revealing a unique ligand recognition mode with the negatively charged head group of LysoPS occupying a polar cavity formed by TM3, 6 and 7, and the hydrophobic tail of LysoPS residing in a lateral open hydrophobic groove formed by TM3-5. Virtual screening and subsequent structural optimization led to the identification of a highly potent and selective antagonist (YL-365). Design of fusion proteins allowed successful determination of the challenging cryo-EM structure of the inactive GPR34 complexed with YL-365, which revealed the competitive binding of YL-365 in a portion of the orthosteric binding pocket of GPR34 and the antagonist-binding-induced allostery in the receptor, implicating the inhibition mechanism of YL-365. Moreover, YL-365 displayed excellent activity in a neuropathic pain model without obvious toxicity. Collectively, this study offers mechanistic insights into the endogenous agonist recognition and antagonist inhibition of GPR34, and provides proof of concept that targeting GPR34 represents a promising strategy for disease treatment.

Molecular mechanism of allosteric modulation for the cannabinoid receptor CB1.

Given the promising clinical value of allosteric modulators of G protein-coupled-receptors (GPCRs), mechanistic understanding of how these modulators alter GPCR function is of significance. Here, we report the crystallographic and cryo-electron microscopy structures of the cannabinoid receptor CB1 bound to the positive allosteric modulator (PAM) ZCZ011. These structures show that ZCZ011 binds to an extrahelical site in the transmembrane 2 (TM2)-TM3-TM4 surface. Through (un)biased molecular dynamics simulations and mutagenesis experiments, we show that TM2 rearrangement is critical for the propagation of allosteric signals. ZCZ011 exerts a PAM effect by promoting TM2 rearrangement in favor of receptor activation and increasing the population of receptors that adopt an active conformation. In contrast, ORG27569, a negative allosteric modulator (NAM) of CB1, also binds to the TM2-TM3-TM4 surface and exerts a NAM effect by impeding the TM2 rearrangement. Our findings fill a gap in the understanding of CB1 allosteric regulation and could guide the rational design of CB1 allosteric modulators.

Community social capital, migration status, and Chinese rural children's psychosocial development.

Migration's impact on Chinese rural children's psychosocial development is the subject of growing research attention. While scholars highlight the critical role of social support, they have yet to systematically examine whether and how community social capital, which provides proximal social support for families, affects rural children's psychosocial development as well as whether such associations vary by children's migration status. Using data from the child component of the 2012 Chinese Urbanization and Labor Migration Survey, this article shows that community social capital reduces children's behavioral and emotional problems; however, left-behind children and migrant children gain less from community social capital than children with at-home parents. In addition, left-behind girls fare worse and gain less from community social capital than left-behind boys. Together, these findings imply that community social capital reinforces the disadvantaged psychosocial development of rural children who experience parental migration and evidence the enduring gender inequality in rural China.

Discovery of 6,7-dihydro-5H-pyrrolo[3,4-d] pyrimidine derivatives as a new class of ATR inhibitors.

Ataxia telangiectasia and Rad3-related (ATR) kinase is a key regulating protein within the DNA damage response (DDR), responsible for sensing replication stress (RS), and has been considered as a potential target for cancer therapy. Herein, we report the discovery of a series of 6,7-dihydro-5H-pyrrolo[3,4-d]-pyrimidine derivatives as a new class of ATR inhibitors. Among them, compound 5g exhibits an IC50 value of 0.007 µM against ATR kinase. In vitro, 5g displays good anti-tumor activity and could significantly reduce the phosphorylation level of ATR and its downstream signaling protein. Overall, this study provides a promising lead compound for subsequent drug discovery targeting ATR kinase.

Discovery of Small Molecule Agonist of Gonadotropin-Releasing Hormone Receptor (GnRH1R).

The gonadotrophin-releasing hormone (GnRH) is a central regulator of the human reproductive system and exerts physiological effects by binding to GnRH1R. The GnRH-GnRH1R system is a promising therapeutic target for the maintenance of reproductive function. There are several GnRH1R agonists on the market, but like GnRH, they are all peptide compounds and are limited by their way of administration (subcutaneous or intramuscular injection). To date, no published GnRH1R small molecule agonists have been reported. In this paper, the HTRF-based screening method has been used to screen our in-house chemical library, and we found and confirmed CD304 as a hit compound. Subsequently, structure optimization led to the discovery of compound 6d, exhibited with a certain GnRH1R activation activity (EC50: 1.59 ± 0.38 µM). Further molecular dynamics simulation experiments showed that 6d can well bind to the orthosteric site of GnRH1R through forming a hydrogen-bonding interaction with Y2836.51. Binding of 6d further induces conformational changes in TM6 and TM7, promoting the formation of a continuous water channel in GnRH1R, thereby promoting GnRH1R activation. This well-characterized hit compound will facilitate the further development of novel small molecule agonists of GnRH1R.

Discovery of 3,6-disubstutited-imidazo[1,2-a]pyridine derivatives as a new class of CLK1 inhibitors.

Inhibition of cdc2-like kinase1 (CLK1) could efficiently induce autophagy and it has been thought as a potential target for treatment of autophagy-related diseases. Herein we report the discovery of a series of 3,6-disubstutited-imidazo[1,2-a]pyridine derivatives as a new class of CLK1 inhibitors. Among them, compound 9e is the most potent one, which exhibits an IC50 value of 4 nM against CLK1 kinase. In vitro, this compound reduces the phosphorylation level of the typical downstream substrates of CLK1 and affects their subcellular redistribution. Further study indicates that 9e is efficient to induce autophagy. Overall, this study provides a promising lead compound for drug discovery targeting CLK1 kinase.

The Early Adhesion Effects of Human Gingival Fibroblasts on Bovine Serum Albumin Loaded Hydrogenated Titanium Nanotube Surface.

The soft tissue sealing at the transmucal portion of implants is vital for the long-term stability of implants. Hydrogenated titanium nanotubes (H2-TNTs) as implant surface treatments were proved to promote the adhesion of human gingival fibroblasts (HGFs) and have broad usage as drug delivery systems. Bovine serum albumin (BSA) as the most abundant albumin in body fluid was crucial for cell adhesion and was demonstrated as a normal loading protein. As the first protein arriving on the surface of the implant, albumin plays an important role in initial adhesion of soft tissue cells, it is also a common carrier, transferring and loading different endogenous and exogenous substances, ions, drugs, and other small molecules. The aim of the present work was to investigate whether BSA-loaded H2-TNTs could promote the early adhesion of HGFs; H2-TNTs were obtained by hydrogenated anodized titanium dioxide nanotubes (TNTs) in thermal treatment, and BSA was loaded in the nanotubes by vacuum drying; our results showed that the superhydrophilicity of H2-TNTs is conducive to the loading of BSA. In both hydrogenated titanium nanotubes and non-hydrogenated titanium nanotubes, a high rate of release was observed over the first hour, followed by a period of slow and sustained release; however, BSA-loading inhibits the early adhesion of human gingival fibroblasts, and H2-TNTs has the best promoting effect on cell adhesion. With the release of BSA after 4 h, the inhibitory effect of BSA on cell adhesion was weakened.

Migration and children's psychosocial development in China: When and why migration matters.

Migration has affected a large number of children in many settings. Despite growing attention to these children, important gaps remain in our understanding of their psychosocial development, as well as the factors that mediate and moderate the impact of migration on children. The present study examines the influences of migration on children's psychosocial well-being in China using a new nationally representative survey. We compared different groups of children age 3-15, including migrant children, left-behind children, and rural and urban children in nonmigrant families. Results show that rural children left behind by both parents were significantly worse off in psychological and behavioral well-being than rural nonmigrant children. By contrast, rural children left behind by one parent and migrant children were no worse off. The disadvantage of left-behind children was mediated by their caregivers' emotional well-being and parenting practices. Frequent contact with migrant parents, but not receipt of remittances, helped ameliorate the vulnerability of left-behind children. These results add to our understanding of how migration affects child development in general.

Prevalence of dental erosion among people with gastroesophageal reflux disease in China.

STATEMENT OF PROBLEM: Gastroesophageal reflux disease (GERD) is typically diagnosed based on symptoms of regurgitation and heartburn, although it may also manifest as asthma-like symptoms, laryngitis, or dental erosion. PURPOSE: The purpose of this prospective, cross-sectional study was to assess the prevalence of dental erosion in people with GERD and to evaluate the association between GERD and dental erosion. MATERIAL AND METHODS: The presence, severity, and pattern of dental erosion was assessed in 51 participants with GERD and 50 participants without GERD using the Smith and Knight tooth wear index. Medical, dietary, and dental histories were collected by questionnaire. Factors potentially related to dental erosion, including GERD, were evaluated by logistic regression. RESULTS: Dental erosion was observed in 31 (60.8%) participants with GERD and 14 (28%) participants without GERD. Bivariate analysis revealed that participants with GERD were more likely to experience dental erosion (crude odds ratio [cOR]: 2.74; 95% CI: 1.19, 6.32) than participants without GERD. Multivariate analysis also revealed that participants with GERD had a higher risk of dental erosion (adjusted odds ratio [aOR]: 3.97; 95% CI: 1.45, 10.89). Consumption of grains and legumes, the most frequently consumed foods in China, did not correlate with dental erosion. However, carbonated beverage consumption was significantly associated with GERD and dental erosion (aOR: 3.34; 95% CI: 1.01, 11.04; P=.04). CONCLUSIONS: GERD was positively correlated with dental erosion. Carbonated beverage consumption can increase the risk of both GERD and dental erosion.

A combination of irsogladine maleate and azithromycin exhibits addictive protective effects in LPS-induced human gingival epithelial cells.

OBJECTIVE: This study aimed to investigate the potential effects of the combination therapy of irsogladine maleate (IM) and azithromycin (AZM) on the inflammation in lipopolysaccharide (LPS)-induced gingival epithelial cells. METHODS: Human gingival epithelial cell OBA-9 was stimulated by LPS to construct the periodontitis model, followed by the treatment of irsogladine maleate (IM) or azithromycin (AZM) with different concentration. Transepithelial electrical resistance (TER) of cells in each group was analyzed, and qRT-PCR and western blotting were used to detect the expressions of inflammatory cytokines. Immunofluorescence staining was performed to detect the protein expression. RESULTS: The TER for cells was significantly decreased while the inflammatory cytokines expressions including IL-6, IL-8, IL-1ß and TNF-α were all significantly increased by LPS compared to the control (P<0.05). However, TER was increased significantly, whereas the cytokine levels were decreased by IM or AZM, but these effects was more apparent in cells treated with IM and AZM combination (P<0.01). Moreover, E-cadherin and vimentin expressions were more positive in the IM and AZM group than in the other groups. The application of ERK and P38 MAPK inhibitors reversed the effects of LPS on cell inflammatory cytokine production and cell TER. CONCLUSION: This study revealed that the combination therapy of IM and AZM performed excellent effects on preventing the inflammatory progression of periodontitis.

[Transplantation of autologous labial salivary glands for severe dry eye].

OBJECTIVE: Autologous labial salivary gland transplantation has been a promising alternative for the treatment of severe dry eye. In this article, we describe the results of the ocular surface changes after labial salivary gland transplantation and investigate the feasibility of this treatment. METHODS: The results of this technique in 8 patients (eyes) who suffered from severe dry eye were prospectively analyzed after surgery (follow-up of 6 months). The best-corrected visual acuity, Schirmer I test, degree of discomfort, usage of pharmaceutical tear substitutes, tear interferometry and slit lamp examination were investigated at different time before and after surgery. RESULTS: All grafts remained viable and the survival rate is 100%. All patients showed significant increase in the Schirmer's test and they expressed great improvement in their ocular discomfort. The use of artificial tear substitutes was reduced because of the increased ocular surface lubrication. CONCLUSION: Although the authors' long-term experience still is limited, we believe that the procedure is a promising alternative approach for severe dry eye.

CHSNet: Automatic lesion segmentation network guided by CT image features for acute cerebral hemorrhage.

Stroke is a cerebrovascular disease that can lead to severe sequelae such as hemiplegia and mental retardation with a mortality rate of up to 40%. In this paper, we proposed an automatic segmentation network (CHSNet) to segment the lesions in cranial CT images based on the characteristics of acute cerebral hemorrhage images, such as high density, multi-scale, and variable location, and realized the three-dimensional (3D) visualization and localization of the cranial lesions after the segmentation was completed. To enhance the feature representation of high-density regions, and capture multi-scale and up-down information on the target location, we constructed a convolutional neural network with encoding-decoding backbone, Res-RCL module, Atrous Spatial Pyramid Pooling, and Attention Gate. We collected images of 203 patients with acute cerebral hemorrhage, constructed a dataset containing 5998 cranial CT slices, and conducted comparative and ablation experiments on the dataset to verify the effectiveness of our model. Our model achieved the best results on both test sets with different segmentation difficulties, test1: Dice = 0.918, IoU = 0.853, ASD = 0.476, RVE = 0.113; test2: Dice = 0.716, IoU = 0.604, ASD = 5.402, RVE = 1.079. Based on the segmentation results, we achieved 3D visualization and localization of hemorrhage in CT images of stroke patients. The study has important implications for clinical adjuvant diagnosis.

Targeting Noncanonical Pyroptosis With a Small Molecular Inhibitor Alleviates Inflammation in the LPS-Induced Keratitis Mouse Model.

Purpose: Pyroptosis, a novel proinflammatory programmed cell death, has been implicated in some ocular diseases. Of special note is the noncanonical pyroptosis that has recently been recognized to play a critical role in microbial keratitis. We previously discovered a new potent small molecular pyroptosis inhibitor, J114. In this investigation, we will explore whether J114 is able to inhibit the noncanonical pyroptosis and the underlying mechanism. Then a lipopolysaccharide (LPS)-induced keratitis mouse model will be used to evaluate the therapeutic effect of J114 in vivo. Methods: In vitro, macrophages originating from humans or mice were stimulated with intracellular LPS to induce noncanonical pyroptosis activation. in vivo, acute keratitis in mouse was induced by LPS intrastromal injection. We verified the protective effect of J114 on noncanonical pyroptosis. Clinical scoring, histological observation, macrophage localization, and quantification of pyroptotic markers in the cornea were used to characterize the therapeutic effects. Results: J114 substantially inhibited the noncanonical pyroptosis and the release of inflammatory cytokines by suppressing the activation of caspase-4/5/11 and the noncanonical NLRP3 inflammasome through blocking the NLRP3-ASC interaction. in vivo, J114 protected against LPS-induced noncanonical pyroptosis of acute keratitis, as manifested by alleviated clinical manifestations and histological disorders, and relieved inflammatory reactions. Conclusions: In this study, we found that J114 could efficiently inhibit LPS-induced noncanonical pyroptosis and revealed the underlying mechanism. This compound displayed significant anti-inflammatory activity in the LPS-induced keratitis mouse model. All the findings indicated that J114 could be a potential lead compound for drug development against inflammatory ocular surface diseases.

Stroke analysis and recognition in functional near-infrared spectroscopy signals using machine learning methods.

Stroke is a high-incidence disease with high disability and mortality rates. It is a serious public health problem worldwide. Shortened onset-to-image time is very important for the diagnosis and treatment of stroke. Functional near-infrared spectroscopy (fNIRS) is a noninvasive monitoring tool with real-time, noninvasive, and convenient features. In this study, we propose an automatic classification framework based on cerebral oxygen saturation signals to identify patients with hemorrhagic stroke, patients with ischemic stroke, and normal subjects. The reflected fNIRS signals were used to detect the cerebral oxygen saturation and the relative value of oxygen and deoxyhemoglobin concentrations of the left and right frontal lobes. The wavelet time-frequency analysis-based features from these signals were extracted. Such features were used to analyze the differences in cerebral oxygen saturation signals among different types of stroke patients and healthy humans and were selected to train the machine learning models. Furthermore, an important analysis of the features was performed. The accuracy of the models trained was greater than 85%, and the accuracy of the models after data augmentation was greater than 90%, which is of great significance in distinguishing patients with hemorrhagic stroke or ischemic stroke. This framework has the potential to shorten the onset-to-diagnosis time of stroke.

Single-Cell RNA-Seq Analysis Identifies Angiotensinogen and Galanin as Unique Molecular Markers of Acinar Cells in Murine Salivary Glands.

The submandibular gland (SMG) and sublingual gland (SLG) are two of three major salivary glands in mammals and comprise serous and mucous acinar cells. The two glands share some functional properties, which are largely dependent on the types of acinar cells. In recent years, while ScRNA-seq (single-cell sequencing) with a 10 × platform has been used to explore molecular markers in salivary glands, few studies have examined the acinar heterogeneity and unique molecular markers between SMG and SLG. This study aimed to identify the molecular markers of acinar cells in the SLG and SMG. We performed ScRNA-seq analyses in 4-week-old mice and verified the screened molecular markers using reverse transcription-quantitative real-time PCR, immunohistochemistry, and immunofluorescence. Our results showed prominently heterogeneous acinar cells, although there was great similarity in the cluster composition between the two glands at 4 weeks. Furthermore, we demonstrated that Agt is a specific marker of SMG serous acinar cells, whereas Gal is a specific marker of SLG mucous acinar cells. Trajectory inference revealed that Agt and Gal represent two types of differential acinar cell clusters during late development in adults. Thus, we reveal previously unknown specific markers for salivary acinar cell diversity, which has extensive implications for their further functional research.

Discovery of 4-(1,2,4-Oxadiazol-5-yl)azepan-2-one Derivatives as a New Class of Cannabinoid Type 2 Receptor Agonists for the Treatment of Inflammatory Pain.

Selectively targeting the cannabinoid receptor CB2 is an attractive therapeutic strategy for the treatment of inflammatory pain without psychiatric side effects mediated by the cannabinoid receptor CB1. Herein, we report the discovery of 4-(1,2,4-oxadiazol-5-yl)azepan-2-one derivatives as a new class of CB2 agonists. Systematic structure-activity relationship investigations resulted in the identification of the most potent compound 25r. This compound displayed high selectivity for CB2 against CB1 (CB2 EC50 = 21.0 nM, Emax = 87%, CB1 EC50 > 30 µM, ratio CB1/CB2 > 1428) with favorable pharmacokinetic properties. Especially, 25r demonstrated significant efficacy in the analgesic model of rodent inflammatory pain. All the results suggest that compound 25r could serve as a lead compound for treating inflammatory pain and deserves further in-depth studies.

Discovery of [1,2,3]Triazolo[4,5-c]quinoline Derivatives as a New Class of Ataxia-Telangiectasia Mutated Kinase Inhibitors.

Ataxia-telangiectasia mutated (ATM) is an atypical serine/threonine protein kinase which is implicated in the repair of DNA double-strand breaks. Numerous reports have shown that ATM inhibition is an attractive target for radiotherapy and chemotherapy sensitization. Herein we report a new series of ATM kinase inhibitors containing the 1H-[1,2,3]triazolo[4,5-c]quinoline scaffold, which was obtained by virtual screening, structural optimization, and structure-activity relationship studies. Among the inhibitors, A011 was one of the most potent, with an IC50 value of 1.0 nM against ATM. In colorectal cancer cells (SW620 and HCT116), A011 was able to inhibit activation of ATM signaling induced by irinotecan (CPT-11) and ionizing radiation and then increased the sensitivity of colorectal cancer cells to irinotecan and ionizing radiation through increasing G2/M arrest and inducing apoptosis. In the SW620 human colorectal adenocarcinoma tumor xenograft model, A011 sensitized SW620 to CPT-11 by inhibiting ATM activity. Collectively, this work has identified a promising lead in the discovery of potent inhibitors against ATM.

Early warning model for death of sepsis via length insensitive temporal convolutional network.

Sepsis is a life-threatening systemic syndrome characterized by various biological, biochemical, and physiological abnormalities. Due to its high mortality, identifying sepsis patients with high risk of in-hospital death early and accurately will help doctors make optimal clinical decisions and reduce the mortality of sepsis patients. In this paper, we propose a length insensitive TCN-based model to predict sepsis patient's death risk in the future k hours, which is the first work for sepsis death risk early warning model only based on vital signs time series to our best knowledge. Furthermore, we design residual connections between temporal residual blocks to improve the prediction performance and stability especially on short input sequences. We validate and evaluate our model on two freely-available datasets, i.e., MIMIC-IV and eICU, from which 16,520 and 29,620 patients are selected respectively. The experiment results show that our model outperforms LSTM and other machine learning methods, as it has the highest sensitivity and Youden index in almost all cases. Meanwhile, the Youden index of the TCN-based model only slightly decreases by 0.0233 and 0.0307 when the time range of the input sequence changes from 24 to 4 h for k equal to 6 and 12, respectively.

HDAC inhibitors improve CRISPR-Cas9 mediated prime editing and base editing.

Recent advances in CRISPR-Cas9 techniques, especially the discovery of base and prime editing, have significantly improved our ability to make precise changes in the genome. We hypothesized that modulating certain endogenous pathway cells could improve the action of those editing tools in mammalian cells. We established a reporter system in which a small fragment was integrated into the genome by prime editing (PE). With this system, we screened an in-house small-molecule library and identified a group of histone deacetylase inhibitors (HDACi) increasing prime editing. We also found that HDACi increased the efficiency of both cytosine base editing (CBE) and adenine base editing (ABE). Moreover, HDACi increased the purity of cytosine base editor products, which was accompanied by an upregulation of the acetylation of uracil DNA glycosylase (UNG) and UNG inhibitor (UGI) and an enhancement of their interaction. In summary, our work demonstrated that HDACi improves Cas9-mediated prime editing and base editing.