The Mechanism of ATP-Dependent Allosteric Protection of Akt Kinase Phosphorylation

SummaryKinases use ATP to phosphorylate substrates; recent findings underscore the additional regulatory roles of ATP. Here, we propose a mechanism for allosteric regulation of Akt1 kinase phosphorylation by ATP. Our 4.7-μs molecular dynamics simulations of Akt1 and its mutants in the ATP/ADP bound/unbound states revealed that ATP occupancy of the ATP-binding site stabilizes the closed conformation, allosterically protecting pT308 by restraining phosphatase access and key interconnected residues on the ATP→pT308 allosteric pathway. Following ATP→ADP hydrolysis, pT308 is exposed and readily dephosphorylated. Site-directed mutagenesis validated these predictions and indicated that the mutations do not impair PDK1 and PP2A phosphatase recruitment. We further probed the function of residues around pT308 at the atomic level, and predicted and experimentally confirmed that Akt1H194R/R273H double mutant rescues pathology-related Akt1R273H. Analysis of classical Akt homologs suggests that this mechanism can provide a general model of allosteric kinase regulation by ATP; as such, it offers a potential avenue for allosteric drug discovery

HIV-1 Membrane-Proximal External Region Fused to Diphtheria Toxin Domain-A Elicits 4E10-Like Antibodies in Mice.

The production of broadly neutralizing antibodies (bNAbs) is a major goal in the development of an HIV-1 vaccine. The membrane-proximal external region (MPER) of gp41, which plays a critical role in the virus membrane fusion process, is highly conserved and targeted by bNAbs 2F5, 4E10, and 10E8. As such, MPER could be a promising epitope for vaccine design. In this study, diphtheria toxin domain A (CRM197, amino acids 1-191) was used as a scaffold to display the 2F5 and 4E10 epitopes of MPER, named CRM197-A-2F5 and CRM197-A-4E10. Modest neutralizing activities were detected against HIV-1 clade B and D viruses in the sera from mice immunized with CRM197-A-4E10. Monoclonal antibodies raised from CRM197-A-4E10 could neutralize several HIV-1 strains, and epitope-mapping analysis indicated that some antibodies recognized the same amino acids as 4E10. Collectively, we show that 4E10-like antibodies can be induced by displaying MPER epitopes using an appropriate scaffold. These results provide insights for HIV-1 MPER-based immunogens design

Risk Coupling Characteristics of Maritime Accidents in Chinese Inland and Coastal Waters Based on N-K Model

The causes of maritime accidents are complex, mostly due to the coupling of four types of factors: human-ship-environmental-management. To effectively analyze the causes of maritime accidents in China, and reveal the risk coupling characteristics of accidents, this paper establishes the N-K model of maritime accident, and calculates and analyzes the four types of coupling of risk factors affecting safety in maritime traffic. This paper collects 922 maritime accidents that occurred in China from 2000 to 2020, and analyzes the location, type, and level of accidents and uses the trigger principle to describe the process of accidents. For marine and inland river accidents, this paper calculates the four types of coupling values of risk factors (single-factor coupling, two-factor coupling, three-factor coupling, four-factor coupling) for comparison and analysis. In addition, this paper calculates the coupling values of six typical maritime accidents of collision, sinking, contact, fire/explosion, stranding, grounding. According to the coupling values and the frequency of sub-factors, this paper analyzes the coupling characteristics of maritime accidents. The results show that in maritime accidents, as the number of risk factors participating in the coupling increases, the coupling value increases, and the multi-factor coupling is more likely to cause accidents. The overall situation of risk coupling causes of maritime accidents is basically consistent with inland river accidents, but they have their own characteristics in the specific degree of risk coupling and the dominant risk elements. In different types of maritime accidents, the risk coupling has different characteristics, and the dominant risk factors are also different

Risk Coupling Characteristics of Maritime Accidents in Chinese Inland and Coastal Waters Based on N-K Model

The causes of maritime accidents are complex, mostly due to the coupling of four types of factors: human-ship-environmental-management. To effectively analyze the causes of maritime accidents in China, and reveal the risk coupling characteristics of accidents, this paper establishes the N-K model of maritime accident, and calculates and analyzes the four types of coupling of risk factors affecting safety in maritime traffic. This paper collects 922 maritime accidents that occurred in China from 2000 to 2020, and analyzes the location, type, and level of accidents and uses the trigger principle to describe the process of accidents. For marine and inland river accidents, this paper calculates the four types of coupling values of risk factors (single-factor coupling, two-factor coupling, three-factor coupling, four-factor coupling) for comparison and analysis. In addition, this paper calculates the coupling values of six typical maritime accidents of collision, sinking, contact, fire/explosion, stranding, grounding. According to the coupling values and the frequency of sub-factors, this paper analyzes the coupling characteristics of maritime accidents. The results show that in maritime accidents, as the number of risk factors participating in the coupling increases, the coupling value increases, and the multi-factor coupling is more likely to cause accidents. The overall situation of risk coupling causes of maritime accidents is basically consistent with inland river accidents, but they have their own characteristics in the specific degree of risk coupling and the dominant risk elements. In different types of maritime accidents, the risk coupling has different characteristics, and the dominant risk factors are also different

The extremity localized classic osteosarcomas have better survival than the axial non-classics

Abstract Background Osteosarcoma is one of the most malignant primary bone cancers, while is rarely reported in China. Of note, very few data of prognosis has been documented in this region. Thus, we carried a retrospective study to identify prognostic factors and to analyze outcomes in patients of both classic and non-classic high-grade osteosarcomas. Classic osteosarcoma is defined as of high-grade histology, age below 40 years, with extremity localized primary tumor, and without detectable metastasis at primary diagnosis. Methods A total of 98 patients (68 classic and 30 non-classic) aged from 4 to 64 years old were diagnosed as high-grade osteosarcoma from 2008 to 2015 in Nanfang Hospital, Guangzhou, China. Univariate and multivariate analyses were performed to identify the independent predictors for overall survival and event-free survival. Kaplan-Meier method was used for survival analysis. Results The median overall survival was 117 vs. 21 months, and the median event-free survival was 31 vs. 6 months in classic and non-classic osteosarcoma, respectively. The most frequently found tumor site was around the knee. The classic osteosarcoma had better overall survival and event-free survival than the non-classics. Tumor site and primary metastasis were found to be associated with overall survival and event-free survival in the univariate analysis. In the multivariate Cox regression analysis, tumor site and primary metastasis were each verified as independent prognostic factors. However, no similar result was found in elevated serum alkaline phosphatase or lactate dehydrogenase. Amputation or limb salvage surgery had no significant effect on overall survival and event-free survival in the extremity osteosarcomas. Classic osteosarcomas with extremity tumor site and free of primary metastasis exhibited better overall survival and event-free survival, while the axial and metastatic non-classics exhibited the worse. Conclusions The extremity classic osteosarcomas have better survivals than the axial non-classic cases. Amputation and limb salvage surgery make no significant change in overall survival and event-free survival in the extremity osteosarcomas. Trial registration Nanfang2013071; Date of registration: 7 September 2013 (retrospectively registered)

Integrated SMRT and Illumina Sequencing Provide New Insights into Crocin Biosynthesis of Gardenia jasminoides

Crocins are valuable bioactive components of gardenia fruit, and their biosynthesis and accumulation have attracted widespread interest. Studies have investigated the biosynthesis and accumulation of crocin based on Illumina sequencing, but there is a lack of reports based on full-length transcriptome sequencing. Utilising SMRT sequencing and high-performance liquid chromatography (HPLC), we explored crocin biosynthesis and accumulation in the fruit of Gardenia jasminoides. HPLC analysis showed that crocins specifically exist in fruit and that the content of crocins increases gradually during fruit development. SMRT sequencing generated 46,715 high-quality full-length isoforms, including 5230 novel isoforms that are not present in the G. jasminoides genome. Furthermore, a total of 46 genes and 91 lncRNAs were involved in the biosynthesis and accumulation of crocin. The qRT-PCR indicated that genes involved in crocin biosynthesis reached a peak in the NOV stage. These findings contributed to our understanding of crocin biosynthesis and accumulation