Rosmarinic acid plus deferasirox inhibits ferroptosis to alleviate crush syndrome-related AKI via Nrf2/Keap1 pathway.

BACKGROUND: Myoglobin (Mb) induced death of renal tubular epithelial cells (RTECs) is a major pathological factor in crush syndrome-related acute kidney injury (CS-AKI). It is unclear whether ferroptosis is involved and could be a target for treatment. PURPOSE: This study aimed to evaluate the potential therapeutic effects of combining the natural small molecule rosemarinic acid (RA) and the iron chelator deferasirox (Dfe) on CS-AKI through inhibition of ferroptosis. METHODS: Sequencing data were downloaded from the GEO database, and differential expression analysis was performed using the R software limma package. The CS-AKI mouse model was constructed by squeezing the bilateral thighs of mice for 16 h with 1.5 kg weight. TCMK1 and NRK-52E cells were induced with 200 µM Mb and then treated with RA combined with Dfe (Dfe + RA, both were 10 µM). Functional and pathological changes in mouse kidney were evaluated by glomerular filtration rate (GFR) and HE pathology. Immunofluorescence assay was used to detect Mb levels in kidney tissues. The expression levels of ACSL4, GPX4, Keap1, and Nrf2 were analyzed by WB. RESULTS: We found that AKI mice in the GSE44925 cohort highly expressed the ferroptosis markers ACSL4 and PTGS2. CS-AKI mice showed a rapid decrease in GFR, up-regulation of ACSL4 expression in kidney tissue, and down-regulation of GPX4 expression, indicating activation of the ferroptosis pathway. Mb was found to deposit in renal tubules, and it has been proven to cause ferroptosis in TCMK1 and NRK-52E cells in vitro. We found that Dfe had a strong iron ion scavenging effect and inhibited ACSL4 expression. RA could disrupt the interaction between Keap1 andNrf2, stabilize Nrf2, and promote its nuclear translocation, thereby exerting antioxidant effects. The combination of Dfe and RA effectively reversed Mb induced ferroptosis in RTECs. CONCLUSION: In conclusion, we found that RA combined with Dfe attenuated CS-AKI by inhibiting Mb-induced ferroptosis in RTECs via activating the Nrf2/Keap1 pathway.

Dynamic Intelligent Scheduling in Low-Carbon Heterogeneous Distributed Flexible Job Shops with Job Insertions and Transfers.

With the rapid development of economic globalization and green manufacturing, traditional flexible job shop scheduling has evolved into the low-carbon heterogeneous distributed flexible job shop scheduling problem (LHDFJSP). Additionally, modern smart manufacturing processes encounter complex and diverse contingencies, necessitating the ability to address dynamic events in real-world production activities. To date, there are limited studies that comprehensively address the intricate factors associated with the LHDFJSP, including workshop heterogeneity, job insertions and transfers, and considerations of low-carbon objectives. This paper establishes a multi-objective mathematical model with the goal of minimizing the total weighted tardiness and total energy consumption. To effectively solve this problem, diverse composite scheduling rules are formulated, alongside the application of a deep reinforcement learning (DRL) framework, i.e., Rainbow deep-Q network (Rainbow DQN), to learn the optimal scheduling strategy at each decision point in a dynamic environment. To verify the effectiveness of the proposed method, this paper extends the standard dataset to adapt to the LHDFJSP. Evaluation results confirm the generalization and robustness of the presented Rainbow DQN-based method.

Protocol for identification and validation of 2'3'-cGAMP-binding proteins by photoaffinity probes.

Mammalian cyclic dinucleotide 2'3'-cGAMP functions as a second messenger in innate immune response. Here, we report a protocol to utilize 2'3'-cGAMP photoaffinity probes to capture 2'3'-cGAMP-binding or 2'3'-cGAMP-interacting proteins from HeLa cell lysate for in-gel visualization by fluorescent imaging or identification by SILAC-based quantitative MS. Further validation is also executed using photoaffinity probes to demonstrate the direct interaction of 2'3'-cGAMP with purified target proteins in vitro or endogenous target proteins in 293T cells. For complete details on the use and execution of this profile, please refer to Hou et al. (2021).

A photoaffinity labeling strategy identified EF1A1 as a binding protein of cyclic dinucleotide 2'3'-cGAMP.

2'3'-cyclic GMP-AMP (2'3'-cGAMP), generated by cyclic GMP-AMP synthase (cGAS) under activation by cytosolic DNA, has a vital role in innate immune response via its receptor protein stimulator of interferon genes (STING) to fight viral infections and tumors. In order to have a complete understanding of biological functions of 2'3'-cGAMP, it is important to find out whether 2'3'-cGAMP has other unrevealed binding proteins present in mammalian cells and executes unknown functions. Here we report the 2'3'-cGAMP-based photoaffinity probes that capture and isolate 2'3'-cGAMP-binding proteins. These probes enable the identification of some potential 2'3'-cGAMP-binding proteins from HeLa cells. EF1A1, an essential protein regulating protein synthesis, is further validated to associate with 2'3'-cGAMP in vitro and in cells to impede protein synthesis. Thus, our studies provide a powerful approach to enable identification of the 2'3'-cGAMP interactome, discover unknown functions of 2'3'-cGAMP, and understand its physiological/pathological roles in tumor immunity and immune-related diseases.

Expression of multiple resistance genes enhances tolerance to environmental stressors in transgenic poplar (Populus × euramericana 'Guariento').

Commercial and non-commercial plants face a variety of environmental stressors that often cannot be controlled. In this study, transgenic hybrid poplar (Populus × euramericana 'Guariento') harboring five effector genes (vgb, SacB, JERF36, BtCry3A and OC-I) were subjected to drought, salinity, waterlogging and insect stressors in greenhouse or laboratory conditions. Field trials were also conducted to investigate long-term effects of transgenic trees on insects and salt tolerance in the transformants. In greenhouse studies, two transgenic lines D5-20 and D5-21 showed improved growth, as evidenced by greater height and basal diameter increments and total biomass relative to the control plants after drought or salt stress treatments. The improved tolerance to drought and salt was primarily attributed to greater instantaneous water use efficiency (WUEi) in the transgenic trees. The chlorophyll concentrations tended to be higher in the transgenic lines under drought or saline conditions. Transformed trees in drought conditions accumulated more fructan and proline and had increased Fv/Fm ratios (maximum quantum yield of photosystem II) under waterlogging stress. Insect-feeding assays in the laboratory revealed a higher total mortality rate and lower exuviation index of leaf beetle [Plagiodera versicolora (Laicharting)] larvae fed with D5-21 leaves, suggesting enhanced insect resistance in the transgenic poplar. In field trials, the dominance of targeted insects on 2-year-old D5-21 transgenic trees was substantially lower than that of the controls, indicating enhanced resistance to Coleoptera. The average height and DBH (diameter at breast height) of 2.5-year-old transgenic trees growing in naturally saline soil were 3.80% and 4.12% greater than those of the control trees, but these increases were not significant. These results suggested that multiple stress-resistance properties in important crop tree species could be simultaneously improved, although additional research is needed to fully understand the relationships between the altered phenotypes and the function of each transgene in multigene transformants.

Identification of expressed sequence tags in an alkali grass (Puccinellia tenuiflora) cDNA library.

Alkali grass (Puccinellia tenuiflora), a monocotyledonous halophyte, can serve as a model of salt tolerance in monocotyledon crops. To elucidate the molecular events associated with salt tolerance in alkali grass, we generated a directional cDNA library from leaves treated with the alkali salt, NaHCO3. Large-scale sequencing of the cDNA library identified 2942 ESTs representing 2366 non-redundant transcripts. These have been deposited in the dbEST division of GenBank. BLASTX evaluation indicated that 1274 of the ESTs were homologous to various known genes/proteins in a broad range of organisms, especially gramineae species. The other 1092 ESTs, which showed little if any homology to known sequences, were considered novel. Based on the encoded proteins, the 1274 identified ESTs fell into 12 functional categories, of which four were abundant, namely metabolism (18.84%), transcription (12.48%), unclassified (11.22%) and cell rescue/defense (9.66%). The 162 unique transcripts corresponding to possible salt-related genes were also identified. This study provides an overview of gene expression in NaHCO3-stressed alkali grass, as well as useful information for further investigation of salt resistance in plants.