Exploring the Structure, Function & Regulation of the Human Glucagon-Like Peptide-1 Receptor

Glucagon-like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion and promotes β-cell function via its receptor (GLP-1R), which therefore is a validated target for the treatment of type 2 diabetes. Due to difficulties with peptide therapeutics, it is important to find small-molecule GLP-1R agonists. This leads to a need to understand the structure, function and regulation of the receptor, particularly, differences between agonisms mediated by GLP-1 (orthosteric agonist) and small molecules. The GLP-1R contains a putative N-terminal signal peptide sequence, which is assessed here by recombinantly expressing several epitope-tagged GLP-1R constructs in HEK293 cells. The findings demonstrate that the GLP-1R is expressed predominately at the plasma membrane and also slightly cytosolic. Only fully glycosylated, mature form of the receptor is able to traffic to the cell surface and performs the function. The signal peptide sequence of the GLP-1R is essential for synthesis. After fulfilling the function, this sequence is cleaved and thus not part of the mature protein. The cleavage of signal peptide is critical for processing and trafficking of the GLP-1R. Based on one of these constructs generated here, a cell line (HEK293: GLP-1R-EGFP) with stable expression of the visible GLP-1R is established, which allows observations and determinations for ligand-mediated receptor internalisation in real time. Compound 2 (6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline) has been described as a GLP-1R allosteric modulator and agonist. Findings here that compound 2-mediated agonisms on both the wild-type (WT) GLP-1R and the mutant with removal of the N-terminal domain provide direct evidence for the allosteric agonism. Interestingly, compound 2-mediated cAMP response is enhanced by orthosteric antagonist exendin 9-39, but the latter inhibits receptor internalisation mediated by compound 2. Recently, it has been hypothesised that the binding of GLP-1 allows a sequence of NRTFD (Asn63-Asp67) in the N-terminus of the GLP-1R to interact with another part of the receptor and cause agonism. This was examined here by generating receptor mutants and synthetic peptides. Findings here that Asp67 plays a key role in stabilising the N-terminal structure of the GLP-1R and thus is critical for processing and trafficking of the receptor protein do not support such hypothesis although synthetic NRTFD mediates a weak and partial agonism on the WTGLP-1R

Salt-Assisted Mechanistic Analysis of Chitosan/Tripolyphosphate Micro- and Nanogel Formation

Self-assembled micro- and nanogels are frequently prepared by mixing tripolyphosphate (TPP) with dilute chitosan solutions. Upon its addition, the TPP ionically cross-links the chitosan molecules into gel-like colloids that range from tens of nanometers to micrometers in diameter. These particles are biocompatible, mucoadhesive and, because they are easy to prepare under very mild conditions, attract widespread interest in the encapsulation of drugs, neutraceuticals, and other bioactive payloads. Despite their broad use, however, their formation mechanism has remained largely obscured by the very fast kinetics of their self-assembly. To this end, we have tuned the TPP and monovalent salt (NaCl) concentrations to dramatically slow down this process (to occur on the time scale of days instead of milliseconds), and then probed the evolution in the size and morphology of micro- and nanogels during their formation. This revealed that the micro- and nanogel formation rates are extremely sensitive to NaCl and TPP concentrations, and that the formation process occurs in two stages: (1) formation of small primary nanoparticles and (2) aggregation of primary particles into larger, higher-order colloids that are obtained at the end of the ionotropic gelation process

Determining the Colloidal Behavior of Ionically Cross-Linked Polyelectrolytes with Isothermal Titration Calorimetry

Mixtures of polyelectrolytes and multivalent counterions can self-assemble into colloidal complexes. These complexes attract widespread interest in applications such as medicine, household product formulations, and separation processes. To facilitate the development of these colloidal dispersions, we examined isothermal titration calorimetry (ITC) as an automated screening tool for identifying the polymer and multivalent counterion compositions that (1) form ionically cross-linked colloidal complexes and (2) lead to their rapid coagulation (and macroscopic phase separation). By studying various polyelectrolyte/multivalent counterion mixtures, we have identified and generalized the features in the ITC data that indicate colloidal complex formation and coagulation. The limitations of this calorimetric screening method were also elucidated. These analyses suggest that ITC can be effective for screening the short-term colloidal behavior of polyelectrolyte/multivalent counterion mixtures but are unreliable in revealing their long-term (equilibrium) properties

Scatter plot of age and volume of testis on the hydrocele side.

Scatter plot of age and volume of testis on the hydrocele side.</p

Data of patient’s age, diagnose time and ultrasound results.

(XLSX)</p

Regression analysis of the moderating effect of entrepreneurial education.

Regression analysis of the moderating effect of entrepreneurial education.</p

Regression analysis of the eastern, central and western regions in China.

Regression analysis of the eastern, central and western regions in China.</p

Scatter plot of age and volume of testis on the normal side.

Scatter plot of age and volume of testis on the normal side.</p

Comparison of the testes’ parameters on the hydrocele and normal sides in 138 patients.

Comparison of the testes’ parameters on the hydrocele and normal sides in 138 patients.</p

Baseline regression.

The integration of rural industries will inevitably lead to new business forms and new models, which put forward new requirements for traditional agricultural finance. The development of digital inclusive finance will provide new momentum for the integration of rural industries. Based on the provincial panel data from 2011 to 2020, the evaluation index system is constructed from three dimensions: industrial integration method, integration subject and integration format, and the development index of rural industrial integration is calculated. This paper establishes double fixed effect model and intermediary effect model to test the effect and path of digital inclusive finance on the integration of rural industries, and further explores the regulatory role and spatial difference of financial support. The results show that: (1) The integration of rural industries shows a growing trend, the eastern region develops more rapidly, while the central and western regions develop more slowly; (2) The digital inclusive finance can promote the integration of rural industries, digitization degree is remarkable, but coverage breadth and using depth are not significant, increasing the rate of per capita electricity consumption and urbanization can promote the integration of rural industries, consumption has limited pulling effect on the integration of rural industries, the per capita investment in fixed assets has no significant effects on the integration of rural industries; (3) The financial availability and the agricultural digitization play a complete intermediary effect; (4) Financial support has a negative moderating effect on the relationship between the two; (5) The eastern and central regions have a significant promoting effect, while the western region has a negative effect.</div