Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria.

Tyrosine phosphorylation is a common protein post-translational modification that plays a critical role in signal transduction and the regulation of many cellular processes. Using a propeptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase-tRNA pair that allows site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray structure of the synthetase reveals a reconfigured substrate-binding site, formed by nonconservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site and determining the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.

Determinants of the Inhibition of DprE1 and CYP2C9 by Antitubercular Thiophenes.

Mycobacterium tuberculosis (Mtb) DprE1, an essential isomerase for the biosynthesis of the mycobacterial cell wall, is a validated target for tuberculosis (TB) drug development. Here we report the X-ray crystal structures of DprE1 and the DprE1 resistant mutant (Y314C) in complexes with TCA1 derivatives to elucidate the molecular basis of their inhibitory activities and an unconventional resistance mechanism, which enabled us to optimize the potency of the analogs. The selected lead compound showed excellent in vitro and in vivo activities, and low risk of toxicity profile except for the inhibition of CYP2C9. A crystal structure of CYP2C9 in complex with a TCA1 analog revealed the similar interaction patterns to the DprE1-TCA1 complex. Guided by the structures, an optimized molecule was generated with differential inhibitory activities against DprE1 and CYP2C9, which provides insights for development of a clinical candidate to treat TB.

Comparison of Typical Alpine Lake Surface Elevation Variations and Different Driving Forces by Remote Sensing Altimetry Method.

Alpine lakes play a significant role in improving watershed ecology, adjusting water storage, and managing regional water resources. They are also a valuable freshwater reservoir, flood storage, and species gene pool in Central Asia. This article validated the accuracy of the CryoSat-2 footprints altimetry dataset for the Lake Bosten and Lake Issyk-Kul ranges. The time series for the surface elevations of the Central Asian alpine lakes Karakul and Chatyrkul were established, based on footprints altimetry data. The lake hydrological drivers were analyzed using remote sensing meteorological reanalysis data of the lake basins. The following main conclusions were reached. The CryoSat-2 footprints altimetry dataset has high confidence in lake surface elevation monitoring. Compared with Hydroweb monitoring results, the agreement between the monitoring results in the range between Lake Bosten and Lake Issyk-Kul are 0.96 and 0.84. The surface elevation of Lake Karakul shows an overall increasing trend with a variation rate of +7.7 cm/yr from 2010 to 2020, which has a positive correlation with the temperature in the basin. This indicates that the increased temperature, which results in the increased snow and ice meltwater in the basin, is the main driving force of the increased lake evolution. The lake surface elevation of Lake Chatyrkul shows an overall decreasing trend, with a variation rate of -9.9 cm/yr from 2010 to 2020, which has a negative correlation with the temperature in the basin. This suggests that Lake Chatyrkul is poorly recharged by snow and ice meltwater. The main driving force of its evolution is the increased evaporative output of the lake due to the increase in temperature. These conclusions prove that temperature and alpine glacial variability within the lake basin play an important role in lake surface elevation variations in alpine regions of Central Asia.

Delivering an mRNA vaccine using a lymphatic drug delivery device improves humoral and cellular immunity against SARS-CoV-2.

The exploration and identification of safe and effective vaccines for the SARS-CoV-2 pandemic have captured the world's attention and remains an ongoing issue due to concerns of balancing protection against emerging variants of concern while also generating long-lasting immunity. Here, we report the synthesis of a novel messenger ribonucleic acid encoding the spike protein in a lipid nanoparticle formulation (STI-7264) that generates robust humoral and cellular immunity following immunization of C57Bl6 mice. In an effort to improve immunity, a clinically focused lymphatic drug delivery device (MuVaxx) was engineered to modulate immune cells at the injection site (epidermis and dermis) and draining lymph node (LN) and tested to measure adaptive immunity. Using MuVaxx, immune responses were elicited and maintained at a 10-fold dose reduction compared to traditional intramuscular (IM) administration as measured by anti-spike antibodies, cytokine-producing CD8 T cells, neutralizing antibodies against the Washington (wild type) strain and South African (Beta) variants, and LN-resident spike-specific memory B cells. Remarkably, a 4-fold-elevated T cell response was observed in MuVaxx-administered vaccination compared to that of IM-administered vaccination. Thus, these data support further investigation into STI-7264 and lymphatic-mediated delivery using MuVaxx for SARS-CoV-2 and VoC vaccines.

Chimeric mRNA-based COVID-19 vaccine induces protective immunity against Omicron and Delta variants.

The emerging SARS-CoV-2 variants of concern (VOCs) exhibit enhanced transmission and immune escape, reducing the effectiveness of currently approved mRNA vaccines. To achieve wider coverage of VOCs, we first constructed a cohort of mRNAs harboring a furin cleavage mutation in the spike (S) protein of predominant VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). The mutation abolished the cleavage between the S1 and S2 subunits. Systematic evaluation in vaccinated mice discovered that individual VOC mRNAs elicited strong neutralizing activity in a VOC-specific manner. In particular, the neutralizing antibodies (nAb) produced by immunization with Beta-Furin and Washington (WA)-Furin mRNAs showed potent cross-reactivity with other VOCs. However, neither mRNA elicited strong neutralizing activity against the Omicron variant. Hence, we further developed an Omicron-specific mRNA vaccine that restored protection against the original Omicron variant and some sublineages. Finally, to broaden the protection spectrum of the new Omicron mRNA vaccine, we engineered an mRNA-based chimeric immunogen by introducing the receptor-binding domain of Delta variant into the entire S antigen of Omicron. The resultant chimeric mRNA induced potent and broadly nAbs against Omicron and Delta, which paves the way to developing new vaccine candidates to target emerging variants in the future.

DeSiphering receptor core-induced and ligand-dependent conformational changes in arrestin via genetic encoded trimethylsilyl 1H-NMR probe.

Characterization of the dynamic conformational changes in membrane protein signaling complexes by nuclear magnetic resonance (NMR) spectroscopy remains challenging. Here we report the site-specific incorporation of 4-trimethylsilyl phenylalanine (TMSiPhe) into proteins, through genetic code expansion. Crystallographic analysis revealed structural changes that reshaped the TMSiPhe-specific amino-acyl tRNA synthetase active site to selectively accommodate the trimethylsilyl (TMSi) group. The unique up-field 1H-NMR chemical shift and the highly efficient incorporation of TMSiPhe enabled the characterization of multiple conformational states of a phospho-ß2 adrenergic receptor/ß-arrestin-1(ß-arr1) membrane protein signaling complex, using only 5 µM protein and 20 min of spectrum accumulation time. We further showed that extracellular ligands induced conformational changes located in the polar core or ERK interaction site of ß-arr1 via direct receptor transmembrane core interactions. These observations provided direct delineation and key mechanism insights that multiple receptor ligands were able to induce distinct functionally relevant conformational changes of arrestin.

Preparation and Characterization of Microencapsulated Ethylenediamine with Epoxy Resin for Self-healing Composites.

Healing agent microcapsules have been used to realize self-healing for polymeric composites. In this work a novel kind of microcapsules encapsulating ethylenediamine (EDA) with epoxy resin as shell material were prepared by interfacial polymerization technology. The oil phase was epoxy resin prepolymer and carbon tetrachloride, and the water phase was EDA and deionized water. Under the action of emulsifier, a stable water-in-oil emulsion was formed. Then the emulsion was added to dimethyl silicone oil, stirred and dispersed, to prepare microcapsules. In addition, the factors affecting the preparation of microcapsules were studied. In this study, Fourier transform infrared(FTIR) was carried out to demonstrate the chemical structure of ethylenediamine microcapsules. Optical microscope(OM) and scanning electron microscope(SEM) were used to observe the morphology of microcapsules. Thermogravimetric analysis and differential scanning calorimetry were done to investigate the thermal properties of microcapsules. Permeability experiment and isothermal aging test were executed to verify the environment resistance of microcapsules. Results showed that EDA was successfully coated in epoxy resin and the microcapsule size was in the range of 50~630 µm. The synthesized microcapsules were thermally stable below 75 °C and perfect permeability resistance to ethanol solvent.