Predicting molecular properties based on the interpretable graph neural network with multistep focus mechanism.

Graph neural networks based on deep learning methods have been extensively applied to the molecular property prediction because of its powerful feature learning ability and good performance. However, most of them are black boxes and cannot give the reasonable explanation about the underlying prediction mechanisms, which seriously reduce people's trust on the neural network-based prediction models. Here we proposed a novel graph neural network named iteratively focused graph network (IFGN), which can gradually identify the key atoms/groups in the molecule that are closely related to the predicted properties by the multistep focus mechanism. At the same time, the combination of the multistep focus mechanism with visualization can also generate multistep interpretations, thus allowing us to gain a deep understanding of the predictive behaviors of the model. For all studied eight datasets, the IFGN model achieved good prediction performance, indicating that the proposed multistep focus mechanism also can improve the performance of the model obviously besides increasing the interpretability of built model. For researchers to use conveniently, the corresponding website (http://graphadmet.cn/works/IFGN) was also developed and can be used free of charge.

Rice ILI atypical bHLH transcription factors antagonize OsbHLH157/OsbHLH158 during brassinosteroid signaling.

Brassinosteroids (BRs) are a group of steroid hormones that play crucial roles in plant growth and development. Atypical bHLH transcription factors that lack the basic region for DNA binding have been implicated in BR signaling. However, the underlying mechanisms of atypical bHLHs in regulation of rice (Oryza sativa) BR signaling are still largely unknown. Here, we describe a systematic characterization of INCREASED LEAF INCLINATION (ILI) subfamily atypical bHLH transcription factors in rice. A total of 8 members, ILI1 to ILI8, with substantial sequence similarity were retrieved. Knockout and overexpression analyses demonstrated that these ILIs play unequally redundant and indispensable roles in BR-mediated growth and development in rice, with a more prominent role for ILI4 and ILI5. The ili3/4/5/8 quadruple and ili1/3/4/7/8 quintuple mutants displayed tremendous BR-related defects with severe dwarfism, erect leaves, and sterility. Biochemical analysis showed that ILIs interact with OsbHLH157 and OsbHLH158, which are also atypical bHLHs and have no obvious transcriptional activity. Overexpression of OsbHLH157 and OsbHLH158 led to drastic BR-defective growth, whereas the osbhlh157 osbhlh158 double mutant developed a typical BR-enhanced phenotype, indicating that OsbHLH157 and OsbHLH158 play a major negative role in rice BR signaling. Further transcriptome analyses revealed opposite effects of ILIs and OsbHLH157/OsbHLH158 in regulation of downstream gene expression, supporting the antagonism of ILIs and OsbHLH157/OsbHLH158 in maintaining the balance of BR signaling. Our results provide insights into the mechanism of BR signaling and plant architecture formation in rice.

Natural allelic variation in a modulator of auxin homeostasis improves grain yield and nitrogen use efficiency in rice.

The external application of nitrogen (N) fertilizers is an important practice for increasing crop production. However, the excessive use of fertilizers significantly increases production costs and causes environmental problems, making the improvement of crop N-use efficiency (NUE) crucial for sustainable agriculture in the future. Here we show that the rice (Oryza sativa) NUE quantitative trait locus DULL NITROGEN RESPONSE1 (qDNR1), which is involved in auxin homeostasis, reflects the differences in nitrate (NO3-) uptake, N assimilation, and yield enhancement between indica and japonica rice varieties. Rice plants carrying the DNR1indica allele exhibit reduced N-responsive transcription and protein abundance of DNR1. This, in turn, promotes auxin biosynthesis, thereby inducing AUXIN RESPONSE FACTOR-mediated activation of NO3- transporter and N-metabolism genes, resulting in improved NUE and grain yield. We also show that a loss-of-function mutation at the DNR1 locus is associated with increased N uptake and assimilation, resulting in improved rice yield under moderate levels of N fertilizer input. Therefore, modulating the DNR1-mediated auxin response represents a promising strategy for achieving environmentally sustainable improvements in rice yield.

Correlation Analysis of Macrophage Distribution and Pathological Features of Carotid Atherosclerotic Plaque.

BACKGROUND: Macrophages play an important role in maintaining the chronic inflammatory of atherosclerosis (AS) and are hallmark of atherosclerotic plaques. They differentiate into different subpopulations under the influence of oxidized lipids and cytokines and play different roles in the formation and development of plaque. To explore the differences in the amount and distribution of different macrophage subpopulations around different carotid plaque pathological features in human AS, and based on these results, to explore the correlation between some macrophage subpopulations and AS pathological features. METHODS: First, we analyzed the single cells RNA-sequence data from the Gene Expression Omnibus DataSets (GSE159677). Second, we investigated the distribution difference of macrophage subpopulations in 61 surgically resected AS plaques by markers staining include CD68, inducible nitric oxide synthase, Arg-1, CD163 and HO-1. RESULTS: The result of single cells RNA-Sequence analysis showed that there were a large number of macrophages infiltrated in AS and they can be categorized into different subpopulations with different transcriptional features and functions; moreover in different part of AS (calcified AS core versus proximal adjacent), the total number and subpopulation ratios were all different. The result of staining analysis showed that macrophages mainly distributed in some pathological lesions such as necrosis, fibrous tissue degeneration, cholesterol crystallization etc., and different subpopulations were distributed differently in these lesions. CONCLUSIONS: This study confirmed that macrophages were heavily infiltrated in atherosclerotic plaques, and there existed subtype variability in different pathological lesions; meanwhile, these results suggested that different macrophage subpopulations may contribute differently in different pathological lesions.

A derivative of trihydroxynaphthalenone and a pyrone metabolite from the endophytic fungus Talaromyces purpurpgenus.

A newly discovered trihydroxynaphthalenone derivative, epoxynaphthalenone (1) involving the condensation of ortho-hydroxyl groups into an epoxy structure, and a novel pyrone metabolite characterized as pyroneaceacid (2), were extracted from Talaromyces purpurpgenus, an endophytic fungus residing in Rhododendron molle. The structures of these compounds were elucidated through a comprehensive analysis of their NMR and HRESIMS data. The determination of absolute configurations was accomplished using electronic circular dichroism (ECD) calculations and CD spectra. Notably, these recently identified metabolites exhibited a moderate inhibitory activity against xanthine oxidase (XOD).

Human epicardial adipose tissue inflammation correlates with coronary artery disease.

BACKGROUND AND AIMS: This study investigates the expression of novel adipocytokines and inflammatory cells infiltration in epicardial adipose tissue (EAT) and subcutaneous adipose tissue (SAT) between 27 coronary artery disease (CAD) and 21 non-CAD (NCAD) patients enrolled from September 2020 to September 2021. METHODS AND RESULTS: Serum, gene, and protein expression levels of the novel adipocytokines were determined using ELISA, RT-qPCR, and western blot analyses. The number of blood vessels and adipocytes morphology were measured via hematoxylin-eosin staining, and inflammatory cells infiltration was examined via immunohistochemistry. Serum ANGPTL8, CTRP5, and Wnt5a levels were higher in the CAD than in the NCAD group, while serum CTRP3, Sfrp5, and ZAG levels were lower in the CAD than in the NCAD group. Compared to the EAT of NCAD and SAT of CAD patients, the EAT of CAD patients had higher mRNA levels of ANGPTL8, CTRP5, and Wnt5a while lower levels of CTRP3, Sfrp5, and ZAG; higher protein expression levels of ANGPTL8 and CTRP5 but lower levels of CTRP3; more blood vessels; and higher infiltration rates of macrophages (CD68 + ), pro-inflammatory M1 macrophages (CD11c + ), mast cells (Tryptase + ), T lymphocytes (CD3 + ), and B lymphocytes (CD20 + ) but lower infiltration rates of anti-inflammatory M2 macrophages (CD206 + ). CONCLUSION: Novel adipocytokines and inflammatory cells infiltration are dysregulated in human EAT, and could be important pathophysiological mechanisms and novelly promising medicating targets of CAD.

Overview of the effects and mechanisms of NO and its donors on biofilms.

Microbial biofilm is undoubtedly a challenging problem in the food industry. It is closely associated with human health and life, being difficult to remove and antibiotic resistance. Therefore, an alternate method to solve these problems is needed. Nitric oxide (NO) as an antimicrobial agent, has shown great potential to disrupt biofilms. However, the extremely short half-life of NO in vivo (2 s) has facilitated the development of relatively more stable NO donors. Recent studies reported that NO could permeate biofilms, causing damage to cellular biomacromolecules, inducing biofilm dispersion by quorum sensing (QS) pathway and reducing intracellular bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) levels, and significantly improving the bactericidal effect without drug resistance. In this review, biofilm hazards and formation processes are presented, and the characteristics and inhibitory effects of NO donors are carefully discussed, with an emphasis on the possible mechanisms of NO resistance to biofilms and some advanced approaches concerning the remediation of NO donor deficiencies. Moreover, the future perspectives, challenges, and limitations of NO donors were summarized comprehensively. On the whole, this review aims to provide the application prospects of NO and its donors in the food industry and to make reliable choices based on these available research results.

Plasma sLRP-1 Level Independently Relates to a Higher Risk of Moderate-Severe Stenosis by Gensini Score in Acute Coronary Syndrome Patients.

Soluble low-density lipoprotein receptor-related protein-1 (sLRP-1) plays a crucial role in facilitating inflammation, lipid accumulation, and atherosclerosis, and the latter factors are involved in the pathology of cardiovascular diseases. This study aimed to explore the ability of plasma sLRP-1 for reflecting stenosis degree in acute coronary syndrome (ACS) patients. sLRP-1 was detected from plasma by enzyme-linked immunosorbent assay in 169 ACS patients and 77 non-ACS subjects (as controls) after admission. Our study illustrated that sLRP-1 was increased in ACS patients versus controls (P < 0.001). Meanwhile, sLRP-1 was positively correlated with body mass index (P = 0.021), white blood cells (P = 0.009), neutrophils (P = 0.002), cardiac troponin I (P = 0.009), and brain natriuretic peptide (P = 0.008) in ACS patients. Notably, sLRP-1 was positively associated with the Gensini score (P = 0.002) and Gensini score stratified stenosis severity (P = 0.004) in ACS patients. After adjustment, sLRP-1 [odds ratio (OR) = 1.333, P = 0.045] independently estimated a higher risk of moderate-severe stenosis, so did numbers of coronary artery lesions (OR = 2.869, P = 0.001), but ejection fraction forecasted a lower risk (OR = 0.880, P = 0.012). Interestingly, a combination of sLRP-1, ejection fraction, and numbers of coronary artery lesions exhibited a good ability to estimate moderate-severe stenosis risk with an area under the curve (95% confidence interval) of 0.845 (0.783-0.906). In summary, increased plasma sLRP-1 represents an aggravated inflammation, impaired cardiac function, and especially a higher stenosis severity in ACS patients.

Identification of CBPA as a New Inhibitor of PD-1/PD-L1 Interaction.

Targeting of the PD-1/PD-L1 immunologic checkpoint is believed to have provided a real breakthrough in the field of cancer therapy in recent years. Due to the intrinsic limitations of antibodies, the discovery of small-molecule inhibitors blocking PD-1/PD-L1 interaction has gradually opened valuable new avenues in the past decades. In an effort to discover new PD-L1 small molecular inhibitors, we carried out a structure-based virtual screening strategy to rapidly identify the candidate compounds. Ultimately, CBPA was identified as a PD-L1 inhibitor with a KD value at the micromolar level. It exhibited effective PD-1/PD-L1 blocking activity and T-cell-reinvigoration potency in cell-based assays. CBPA could dose-dependently elevate secretion levels of IFN-γ and TNF-α in primary CD4+ T cells in vitro. Notably, CBPA exhibited significant in vivo antitumor efficacy in two different mouse tumor models (a MC38 colon adenocarcinoma model and a melanoma B16F10 tumor model) without the induction of observable liver or renal toxicity. Moreover, analyses of the CBPA-treated mice further showed remarkably increased levels of tumor-infiltrating CD4+ and CD8+ T cells and cytokine secretion in the tumor microenvironment. A molecular docking study suggested that CBPA embedded relatively well into the hydrophobic cleft formed by dimeric PD-L1, occluding the PD-1 interaction surface of PD-L1. This study suggests that CBPA could work as a hit compound for the further design of potent inhibitors targeting the PD-1/PD-L1 pathway in cancer immunotherapy.

Biological Evaluation of 8-Methoxy-2,5-dimethyl-5H-indolo[2,3-b] Quinoline as a Potential Antitumor Agent via PI3K/AKT/mTOR Signaling.

Chemotherapy is commonly used clinically to treat colorectal cancer, but it is usually prone to drug resistance, so novel drugs need to be developed continuously to treat colorectal cancer. Neocryptolepine derivatives have attracted a lot of attention because of their good cytotoxic activity; however, cytotoxicity studies on colorectal cancer cells are scarce. In this study, the cytotoxicity of 8-methoxy-2,5-dimethyl-5H-indolo[2,3-b] quinoline (MMNC) in colorectal cells was evaluated. The results showed that MMNC inhibits the proliferation of HCT116 and Caco-2 cells, blocks the cell cycle in the G2/M phase, decreases the cell mitochondrial membrane potential and induces apoptosis. In addition, the results of western blot experiments suggest that MMNC exerts cytotoxicity by inhibiting the expression of PI3K/AKT/mTOR signaling pathway-related proteins. Based on these results, MMNC is a promising lead compound for anticancer activity in the treatment of human colorectal cancer.

[Autosomal dominant mental retardation type 5 caused by SYNGAP1 gene mutations: a report of 8 cases and literature review]. / SYNGAP1åŸºå› å˜å¼‚ç›¸å ³å¸¸æŸ“è‰²ä½“æ˜¾æ€§æ™ºåŠ›éšœç¢5型8ä¾‹å¹¶æ–‡çŒ®å¤ä¹ .

OBJECTIVES: To summarize the clinical phenotype and genetic characteristics of children with autosomal dominant mental retardation type 5 caused by SYNGAP1 gene mutations. METHODS: A retrospective analysis was performed on the medical data of 8 children with autosomal dominant mental retardation type 5 caused by SYNGAP1 gene mutations who were diagnosed and treated in the Department of Pediatrics, Xiangya Hospital of Central South University. RESULTS: The mean age of onset was 9 months for the 8 children. All children had moderate-to-severe developmental delay (especially delayed language development), among whom 7 children also had seizures. Among these 8 children, 7 had novel heterozygous mutations (3 with frameshift mutations, 2 with nonsense mutations, and 2 with missense mutations) and 1 had 6p21.3 microdeletion. According to the literature review, there were 48 Chinese children with mental retardation caused by SYNGAP1 gene mutations (including the children in this study), among whom 40 had seizures, and the mean age of onset of seizures was 31.4 months. Frameshift mutations (15/48, 31%) and nonsense mutations (19/48, 40%) were relatively common in these children. In terms of treatment, among the 33 children with a history of epileptic medication, 28 (28/33, 85%) showed response to valproic acid antiepileptic treatment and 16 (16/33, 48%) achieved complete seizure control after valproic acid monotherapy or combined therapy. CONCLUSIONS: Children with autosomal dominant mental retardation type 5 caused by SYNGAP1 gene mutations tend to have an early age of onset, and most of them are accompanied by seizures. These children mainly have frameshift and nonsense mutations. Valproic acid is effective for the treatment of seizures in most children.

Oxidant stress-sensitive circRNA Mdc1 controls cardiomyocyte chromosome stability and cell cycle re-entry during heart regeneration.

Targeting cardiomyocyte plasticity has emerged as a new strategy for promoting heart repair after myocardial infarction. However, the precise mechanistic network underlying heart regeneration is not completely understood. As noncoding RNAs, circular RNAs (circRNAs) play essential roles in regulating cardiac physiology and pathology. The present study aimed to investigate the potential roles of circMdc1 in cardiac repair after injury and elucidate its underlying mechanisms. Here, we identified that circMdc1 levels were upregulated in postnatal mouse hearts but downregulated in the regenerative myocardium. The expression of circMdc1 in cardiomyocytes is sensitive to oxidative stress, which was attenuated by N-acetyl-cysteine. Enforced circMdc1 expression inhibited cardiomyocyte proliferation, while circMdc1 silencing led to cardiomyocyte cell cycle re-entry. In vivo, the cardiac-specific adeno-associated virus-mediated knockdown of circMdc1 promoted cardiac regeneration and heart repair accompanied by improved heart function. Conversely, circMdc1 overexpression blunted the regenerative capacity of neonatal hearts after apex resection. Moreover, circMdc1 was able to block the translation of its host gene Mdc1 specifically by binding to PABP, affecting DNA damage and the chromosome stability of cardiomyocytes. Furthermore, overexpression of Mdc1 caused damaged mouse hearts to regenerate and repair after myocardial infarction in vivo. Oxidative stress-sensitive circMdc1 plays an important role in cardiac regeneration and heart repair after injury by regulating DNA damage and chromosome stability in cardiomyocytes by blocking the translation of the host gene Mdc1.

Rh(III)-Catalyzed One-Pot Three-Component Diannulation of Benzils, Ammonium Acetate, and Alkynes to Build 1,1'-Biisoquinolines.

An efficient one-pot synthesis of 1,1'-biisoquinolines by a three-component reaction of commercially available raw materials (benzils, NH4OAc, and alkynes) is disclosed. This complicated reaction involves in situ diimine formation via benzil-NH4OAc condensation, Rh(III)-catalyzed 2-fold imine-directed C-H activation, and annulation with alkynes. Both symmetric and unsymmetric 1,1'-biisoquinolines could be assembled in moderate to high yields. The reaction mechanism is supported through ESI-MS, in which Cp*ClRh+, Cp*(OAc)Rh+, Cp*(OPiv)Rh+, and two rhodacycle intermediates are successfully detected to explain the evolution of rhodium species.

Facile assembly and excellent elimination behavior of porous BiOBr-g-C3N4 heterojunctions for organic pollutants.

Photocatalysis can be an effective technique for eliminating organic contaminants from water. In this study, BiOBr flower-spheres coupled with porous graphite carbon nitride (g-C3N4) were synthesized by controlling the dosage of cetyltrimethylammonium bromide (CTAB). Various characterization techniques were then applied to elucidate the structure-performance relationships of the resulting heterojunction photocatalysts in degrading organic dyes. Experimental results established an optimal molar ratio for KBr to CTAB of 5:1. Benefiting from a remarkable porous structure and tight coupling between porous g-C3N4 and BiOBr, the optimal BiOBr-g-C3N4(2%) exhibited enhanced visible light absorption capability and promoted the separation of photoinduced carriers. Total removal efficiency for rhodamine B (RhB, 25.0 mL, 20.0 mg L-1) reached 87% within 30 min in the presence of BiOBr-g-C3N4(2%) (20.0 mg) (i.e., 1.51 µmol (gphotocatalyst min)-1), which is superior to the performance of BiOBr (72%) (i.e., 1.25 µmol (gphotocatalyst min)-1), g-C3N4 (21%) (i.e., 0.37 µmol (gphotocatalyst min)-1). Furthermore, the photocatalytic reaction rate constant over the optimal heterojunction was 0.034 min-1, which is significantly larger than those of porous g-C3N4 (0.003 min-1) and BiOBr (0.015 min-1). Moreover, this type II heterojunction showed good universality for other organic dyes (such as methyl violet, methylene blue, and crystal violet), highlighting a promising potential role in the elimination of environmental pollutants.

Light Emitting Diodes Irradiation Regulates miRNA-877-3p to Promote Cardiomyocyte Proliferation.

Mammalian cardiomyocytes (CMs) maintain a low capacity for self-renewal in adulthood, therefore the induction of CMs cycle re-entry is an important approach to promote myocardial repair after injury. Recently, photobiomodulation (PBM) has been used to manipulate physiological activities of various tissues and organs by non-invasive means. Here, we demonstrate that conditioned PBM using light-emitting diodes with a wavelength of 630 nm (LED-Red) was capable of promoting the proliferation of neonatal CMs. Further studies showed that low-power LED-Red affected the expression of miR-877-3p and promoted the proliferation of CMs. In contrast, silencing of miR-877-3p partially abolished the pro-proliferative actions of LED-Red irradiation on CMs. Mechanistically, GADD45g was identified as a downstream target gene of miR-877-3p. Conditioned LED-Red irradiation also inhibited the expression of GADD45g in neonatal CMs. Moreover, GADD45g siRNA reversed the positive effect of LED-Red on the proliferation of neonatal CMs. Taken together, conditioned LED-Red irradiation increased miR-877-3p expression and promoted the proliferation of neonatal CMs by targeting GADD45g. This finding provides a new insight into the role of LED-Red irradiation in neonatal CMs biology and suggests its potential application in myocardial injury repair.

Design, synthesis, and cytotoxic activities of isaindigotone derivatives as potential anti-gastric cancer agents.

A series of novel derivatives of isaindigotone, which comes from the root of isaits indinatca Fort, were synthesised (Compound 1-26). Four human gastrointestinal cancer cells (HCT116, PANC-1, SMMC-7721, and AGS) were employed to evaluate the anti-proliferative activity. Among them, Compound 6 displayed the most effective inhibitory activity on AGS cells with an IC50 (50% inhibitory concentration) value of 2.2 µM. The potential mechanism study suggested that Compound 6 induced apoptosis in AGS cells. The collapse of mitochondrial membrane potential (MMP) in AGS cells was proved. In docking analysis, good affinity interaction between Compound 6 and AKT1 was discovered. Treatment of AGS cells with Compound 6 also resulted in significant suppression of PI3K/AKT/mTOR signal pathway. The collapse of MMP and suppression of PI3K/AKT/mTOR signal pathway may be responsible for induction of apoptosis. This derivative Compound 6 could be useful as an underlying anti-tumour agent for treatment of gastric cancer.

Wideband signal detection in multipath environment affected by impulsive noise.

Detecting the presence or absence of a known signal is an important aspect of underwater acoustic (UWA) communications as it is a vital first step to process the received data. The influence of impulsive noise and multipath propagation on detection are both considered in this paper. Many robust detectors have been designed to cope with impulsive noise, but they tend to ignore the impact of multipath propagation, which may decrease the detection probability. To emphasize the role of multipath channel, we propose two-stage detection methods. In the first stage, the channel is estimated based on robust orthogonal matching pursuit, while in the second stage, detectors that use the channel estimation from the first stage, namely augmented log-likelihood ratio detector and augmented pseudo-correlation detector, are developed. The former detector is parametric and the latter one is non-parametric. The improved performance is demonstrated in simulated multipath channel and actual UWA channel with both simulated noise and recorded snapping shrimp noise, indicating that the proposed detectors are resistant to impulsive noise. It is also shown that the use of multipath information helps to improve the detection performance.

A Novel Isaindigotone Derivative Displays Better Anti-Proliferation Activities and Induces Apoptosis in Gastric Cancer Cells.

Isaindigotone is an alkaloid containing a pyrrolo-[2,1-b]quinazoline moiety conjugated with a benzylidene group and isolated from the root of Isatis indigotca Fort. However, further anticancer activities of this alkaloid and its derivatives have not been fully explored. In this work, a novel isaindigotone derivative was synthesized and three different gastric cell lines and one human epithelial gastric cell line were used to study the anti-proliferation effects of the novel isaindigotone derivative BLG26. HGC27 cells and AGS cells were used to further explore the potential mechanisms. BLG26 exhibited better anti-proliferation activities in AGS cells with a half-maximal inhibitory concentration (IC50) of 1.45 µM. BLG26 caused mitochondrial membrane potential loss and induced apoptosis in both HGC27 cells and AGS cells by suppressing mitochondrial apoptotic pathway and PI3K/AKT/mTOR axis. Acute toxicity experiment showed that LD50 (median lethal dose) of BLG26 was above 1000.0 mg/kg. This research suggested that BLG26 can be a potential candidate for the treatment of gastric cancer.

Design, Synthesis and Biological Evaluation of Neocryptolepine Derivatives as Potential Anti-Gastric Cancer Agents.

Natural products play an important role in drug development and lead compound synthesis. Neocryptolepine is a polycyclic quinoline compound isolated from Cryptolepis sanguinolent. The cytotoxicity of neocryptolepine to gastric cancer cells AGS, MKN45, HGC27, and SGC7901 was not very strong, and it also had certain toxicity to gastric mucosa cells GES-1. Therefore, a series of neocryptolepine derivatives were synthesized by the modification of the structure of neocryptolepine, and their cytotoxicity was evaluated. The results showed that compounds C5 and C8 exhibited strong cytotoxicity to AGS cells. The cell colony formation and cell migration experiments suggested that compounds C5 and C8 could inhibit the proliferation and cell migration of AGS and HGC27 cells. Cell cycle and apoptosis experiments showed that compounds C5 and C8 did not cause the apoptosis of AGS and HGC27 cells but, mainly, caused cell necrosis. Compound C5 had no significant effect on AGS and HGC27 cell cycles at low concentration. After treatment with AGS cells for 24 h at high concentration, compound C5 could significantly arrest the AGS cell cycle in the G2/M phase. Compound C8 had no significant effect on the AGS and HGC27 cell cycles. The results of molecular docking and Western blot showed that compounds C5 and C8 might induce cytotoxicity through the PI3K/AKT signaling pathway. Therefore, compounds C5 and C8 may be promising lead compounds for the treatment of gastric cancer.

N-glycosylation of the human neuropeptide QRFP receptor (QRFPR) is essential for ligand binding and receptor activation.

The newly identified pyroglutamylated RFamide peptide (QRFP) signaling system has been shown to be implicated in regulating a variety of physiological processes. G-protein-coupled receptors (GPCRs) are preferentially N-glycosylated on extracellular domains. The human QRFP receptor QRFPR (GPR103) possesses three N-glycosylation consensus sites, two located on the N-terminal domain (N5 and N19) and one on the first extracellular loop (ECL1) (N106); however, to date, their role in QRFPR expression and signaling has not been established. Here, we combined mutants with glutamine substitution of the critical asparagines of the consensus sites with glycosidase PNGase F and N-glycosylation inhibitor tunicamycin to study the effect of N-glycosylation in the regulation of QRFPR cell surface expression and signaling. Western blot analysis performed with site-directed mutagenesis revealed that two asparagines at N19 in the N-terminus and N106 in ECL1, but not N5 in the N-terminus, served as sites for N-glycosylation. Treatment with PNGase F and tunicamycin resulted in a reduction in both two-protein species, ~43 kDa and ~85 kDa in size, by 2-4 kDa. Analysis with confocal microscopy and quantitative ELISA showed that N-glycosylation of QRFPR is not essentially required for targeting the cell membrane. However, further binding assay and functional assays demonstrated that removal of N-glycosylation sequons or treatment with tunicamycin led to significant impairments in the interaction of receptor with QRFP26 and downstream signaling. Thus, our findings suggest that for the human QRFP receptor (QRFPR), N-glycosylation is not important for cell surface expression but is a pre-requisite for ligand binding and receptor activation.