Molecular features of the ligand-free GLP-1R, GCGR and GIPR in complex with Gs proteins.

Class B1 G protein-coupled receptors (GPCRs) are important regulators of many physiological functions such as glucose homeostasis, which is mainly mediated by three peptide hormones, i.e., glucagon-like peptide-1 (GLP-1), glucagon (GCG), and glucose-dependent insulinotropic polypeptide (GIP). They trigger a cascade of signaling events leading to the formation of an active agonist-receptor-G protein complex. However, intracellular signal transducers can also activate the receptor independent of extracellular stimuli, suggesting an intrinsic role of G proteins in this process. Here, we report cryo-electron microscopy structures of the human GLP-1 receptor (GLP-1R), GCG receptor (GCGR), and GIP receptor (GIPR) in complex with Gs proteins without the presence of cognate ligands. These ligand-free complexes share a similar intracellular architecture to those bound by endogenous peptides, in which, the Gs protein alone directly opens the intracellular binding cavity and rewires the extracellular orthosteric pocket to stabilize the receptor in a state unseen before. While the peptide-binding site is partially occupied by the inward folded transmembrane helix 6 (TM6)-extracellular loop 3 (ECL3) juncture of GIPR or a segment of GCGR ECL2, the extracellular portion of GLP-1R adopts a conformation close to the active state. Our findings offer valuable insights into the distinct activation mechanisms of these three important receptors. It is possible that in the absence of a ligand, the intracellular half of transmembrane domain is mobilized with the help of Gs protein, which in turn rearranges the extracellular half to form a transitional conformation, facilitating the entry of the peptide N-terminus.

Effects of exogenous abscisic acid on oil content, fatty acid composition, biodiesel properties and lipid components in developing Siberian apricot (Prunus sibirica) seeds.

Previous studies in Siberian apricot (Prunus sibirica) seed kernel (SASK) have suggested the involvement of abscisic acid (ABA) signaling pathway in oil accumulation. However, there are few reports on the effects of ABA on the metabolism of fatty acids (FA) in seed development. Here, we first evaluated the response of developing SASK to ABA treatment, with a focus on oil content, FA composition, biodiesel properties, lipid compounds and gene expressions. Compared with control samples, the application of exogenous ABA increased the total oil content by 6.55% in mature SASK. The C18:1 content markedly increased in ABA treatment, and conversely C16:0 decreased. Exogenous ABA also improved the biodiesel properties of SASK oil, making it better suited to the specifications of biodiesel standards. Furthermore, the molecular species of phosphatidylcholine (PC), phosphatidic acid (PA), diacylglycerol (DAG) and triacylglycerol (TAG) were detected using lipidomics analysis. The 18:1/18:1 was the main component in PA, PC and DAG, while the main components of 18:1/18:1/18:2, 18:1/18:1/18:3, 18:2/18:2/18:2 and 18:1/18:1/18:1 in TAG. Most lipid species gradually increased with SASK maturity. In addition, the relative contents of TAG-18:1/18:1/18:2 and TAG-18:1/18:1/18:1 in developing SASK increased with the application of exogenous ABA. We also detected elevated gene expression of key genes involved in ABA chemical pathway, which likely affected FA biosynthesis and accumulation. Our results provide insight into the effects of ABA on the oil accumulation in developing SASK, which has direct applications to improving the quality of SASK-derived biodiesel.

Genome-wide characterization of the NUCLEAR FACTOR-Y (NF-Y) family in Citrus grandis identified CgNF-YB9 involved in the fructose and glucose accumulation.

BACKGROUND: Nuclear factor Y (NF-Y) is increasingly known to be involved in many aspects of plant growth and development. To date, the systematic characterization of NF-Y family has never been reported in Citrus grandis. OBJECTIVE: Genome-wide characterization of C. grandis NF-Y (CgNF-Y) family and analysis of their role in sucrose metabolism. METHODS: NF-Y conserved models were employed to identify CgNF-Y genes from genomic data. Phylogenetic tree was generated by the neighbor-joining method using program MEGA 7.0. Based on our previous transcriptomic data, the transcription levels were calculated by RSEM software and were clustered by ShortTime-series Expression Miner. The plant expression vector of CgNF-YB9 was constructed using In-Fusion Cloning and transferred into tobacco by leaf disc transformation method. Soluble sugars and gene expressions were analysis by HPLC and qRT-PCR, respectively. RESULTS: A total of 24 CgNF-Y genes (6 CgNF-YAs, 13 CgNF-YBs and 5 CgNF-YCs) were identified with conserved domains. Phylogenetic analysis of the NF-Y proteins indicated that NF-YA, NF-YB and NF-YC could be categorized into four, five and three clades, respectively. Expression profiling analysis reflected spatio-temporally distinct expression patterns for CgNF-Y genes. Importantly, we observed a positive correlation between the expression level of CgNF-YB9 and the content of soluble sugar. Moreover, CgNF-YB9-corelated genes were enriched in carbohydrate metabolism. In CgNF-YB9 overexpression lines, sucrose content showed a decrease, whereas glucose and fructose contents displayed an increase. As expected, the transcription levels of sucrose-phosphate synthase and vacuolar invertase in transgenic Line 3 were observed with significantly down- and up-regulated, respectively. CONCLUSIONS: The structure, phylogenetic relationship and expression pattern of 24 CgNF-Y genes were identified, and CgNF-YB9 was involved in sucrose metabolism.

WRINKLED1 transcription factor orchestrates the regulation of carbon partitioning for C18:1 (oleic acid) accumulation in Siberian apricot kernel.

WRINKLED1 (WRI1), an APETALA2 (AP2)-type transcription factor, has been shown to be required for the regulation of carbon partitioning into fatty acid (FA) synthesis in plant seeds. To our knowledge, the regulatory network of WRI1 remains unknown in Prunus sibirica kernel (PSK), a novel woody biodiesel feedstock in China. In this study, based on the transcriptional data from developing oilseeds of multiple plant species, we identified 161 WRI1-coexpressed genes using weighted gene co-expression network analysis (WGCNA). The major portion of WRI1-coexpressed genes was characterized to be involved in carbon partitioning and FA biosynthesis. Additionally, we detected the temporal patterns for oil content and FA compositions in developing PSK from two different germplasms (AS-85 and AS-86). The major differences between the two germplasms are higher contents of oil and C18:1 in AS-85 than in AS-86 at a mature stage. Thus, AS-85 and AS-86 are desirable materials to explore the molecular and metabolic mechanisms of oil accumulation in Siberian apricot. Expression analysis in developing PSK of AS-85 and AS-86 indicated that the expression level of P. sibirica WRI1 (PsWRI1) was closely correlated to accumulative rate of oil. Also, the comparison of expression profiles in developing PSK of AS-85 and AS-86 displayed that the pPK, E1-α, E2, TAL, BC, MCMT, BS, SAD and FAD2 have a high correlation with PsWRI1. Transient expression showed that ProSAD- and ProBS-driving GUS expression showed no substantial difference between AS-85 and AS-86, while the expression level of ProPEPCK-AS-85 driving GUS was significantly higher than that of ProPEPCK-AS-86 driving GUS. Additionally, transient co-transformation with PsWRI1 revealed that ProSAD, ProPEPCK and ProBS activity could be specifically up-regulated by PsWRI1. This regulatory mechanism of PsWRI1 may create a steep concentration difference, thereby facilitating carbon flux into C18:1 accumulation in developing PSK. Overall, all our findings imply a versatile mechanism of WRI1 to optimize carbon allocation for oil accumulation, which can provide reference for researching the woody biodiesel plants.

Fruit characteristics, soluble sugar compositions and transcriptome analysis during the development of Citrus maxima "seedless", and identification of SUS and INV genes involved in sucrose degradation.

Citrus maxima "seedless" is originally from Malaysia, and now is widely cultivated in Hainan province, China. The essential features of this cultivar are thin skin, green epicarp and seedless at the ripening stage. Here, using C. maxima "seedless" as experimental material, we investigated the physical and inclusion indicators, and found the accumulation of storage compounds during 120-210 DAF leading to inconsistent increase between volume and weight. Component analysis of soluble sugar indicated that arabinose and xylose have a high content in early development of pummelo juice sacs (PJS), whereas fructose, glucose and sucrose show a significant increase during PJS maturation. To clarify a global overview of the gene expressing profiles, the PJSs from four periods (60, 120, 180 and 240 DAF) were selected for comparative transcriptome analysis. The resulting 8275 unigenes showed differential expression during PJS development. Also, the stability of 11 housekeeping genes were evaluated by geNorm method, resulting in a set of five genes (UBC, ACT, OR23, DWA2 and CYP21D) used as control for normalization of gene expression. Based on transcriptome data, 5 sucrose synthases (SUSs) and 10 invertases (INVs) were identified to be involved in sucrose degradation. Importantly, SUS4 may be responsible for arabinose and xylose biosynthesis to form the cell wall in early development, while SUS3 and VIN2 may be important in the accumulation of soluble hexose leading to cell expansion through an osmotic-independent pathway in late development. The information provides valuable metabolite and genetic resources in C. maxima "seedless", and is important for achieving high fruit yield and quality.

Decision tree based diagnostic system for moderate to severe obstructive sleep apnea.

One of the major modern medical issues, obstructive sleep apnea (OSA), particularly at moderate to severe levels, may potentially cause cardiovascular morbidity and mortality. However, polysomnography (PSG), a gold standard tool in diagnosing OSA, is cumbersome, has limited availability, and is costly and time-consuming. Clinical prediction models thus are absolutely necessary in screening patients with OSA. Furthermore, the performance of the published prediction formulas is not satisfactory for Chinese populations. The aim of this study was to develop and validate a simple and accurate prediction system for the diagnosis of moderate to severe OSA by integrating an expert-based feature extraction technique with decision tree algorithms which have automatic feature selection capability in screening the moderate to severe OSA cases in Taiwan. Moreover, the backward stepwise multivariable logistic regression model and four other decision tree algorithms were also employed for comparison. The results showed that the proposed best prediction formula, with an overall accuracy reaching to 96.9 % in sensitivity = 98.2 % and specificity = 93.2 %, could present a good tool for screening moderate and severe Taiwanese OSA patients who require further PSG evaluation and medical intervention. Results also indicate that the proposed best prediction formula is simple, accurate, and reliable, and outperforms all the other prediction formulae considered in the present study. The proposed clinical prediction formula derived from three non-invasive features (Sex, Age, and AveSBP) may help prioritize patients for PSG studies as well as avoid a diagnosis of PSG in subjects who have a low probability of having the disease.