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Hirudin variants are the most powerful thrombin inhibitors discovered to date, with a lower risk of bleeding than heparin. For anticoagulation, the C-termini of hirudin variants bind to the exocite I of thrombin. Anticoagulant effects of gene-recombinant hirudin are weaker than natural hirudin for the reason of lacking tyrosine O-sulfation at C-terminus.An integrative pharmacological study was carried out using molecular dynamic, molecular biological and in vivo and in vitro experiments to elucidate the anticoagulant effects of protein-engineered hirudins.Molecular dynamic analysis showed that modifications of the C-termini of hirudin variant 1 of Hirudo medicinalis (HV1) and hirudin variant 2 of Hirudinaria manillensis (HM2) changed the binding energy of the C-termini to human thrombin. The study indicated that Asp61 of HM2 that corresponds to sulfated Tyr63 of HV1 is critical for inhibiting thrombin activities. Further, the anticoagulant effects of HV1 and HM2 were improved when the amino acid residues adjacent to Asp61 were mutated to Asp. These improvements were prolongation of the activated partial thromboplastin time, prothrombin time and thrombin time of human blood, and decreased K

The major pathological feature of Alzheimer’s disease (AD) is the aggregation of amyloid β peptide (Aβ) in the brain. Inhibition of Aβ42 aggregation may prevent the advancement of AD. This study employed molecular dynamics, molecular docking, electron microscopy, circular dichroism, staining of aggregated Aβ with ThT, cell viability, and flow cytometry for the detection of reactive oxygen species (ROS) and apoptosis. Aβ42 polymerizes into fibrils due to hydrophobic interactions to minimize free energy, adopting a β-strand structure and forming three hydrophobic areas. Eight dipeptides were screened by molecular docking from a structural database of 20 L-α-amino acids, and the docking was validated by molecular dynamics (MD) analysis of binding stability and interaction potential energy. Among the dipeptides, arginine dipeptide (RR) inhibited Aβ42 aggregation the most. The ThT assay and EM revealed that RR reduced Aβ42 aggregation, whereas the circular dichroism spectroscopy analysis showed a 62.8% decrease in β-sheet conformation and a 39.3% increase in random coiling of Aβ42 in the presence of RR. RR also significantly reduced the toxicity of Aβ42 secreted by SH-SY5Y cells, including cell death, ROS production, and apoptosis. The formation of three hydrophobic regions and polymerization of Aβ42 reduced the Gibbs free energy, and RR was the most effective dipeptide at interfering with polymerization.

Manihot esculenta Crantz (cassava) is an important crop with high photosynthetic rate and high yield. It is classified as a C3-C4 plant based on its photosynthetic and structural characteristics. To investigate the structural and photosynthetic characteristics of cassava leaves at the cellular level, a single-cell transcriptome atlas of cassava leaves was created. A total of 11,177 high-quality leaf cells were divided into 15 cell clusters. Based on leaf cell marker genes, we identified three major tissues of cassava leaves, which were mesophyll, epidermis, and vascular tissue, and analyzed their distinctive properties and metabolic activity. To supplement the genes for identifying the types of leaf cells, we screened 120 candidate marker genes. We constructed a leaf cell development trajectory map and discovered six genes related to cell differentiation fate. The structural and photosynthetic properties of cassava leaves analyzed at the single cellular level provide a theoretical foundation for further enhancing cassava yield and nutrition.

Yechun Pei,1– 3,* Zhengpan Xiao,1,2,* Shuangshuang Wei,1,2 Meiqi Peng,1,2 Chenghui Luo,1,2 Dayong Wang2,3 1School of Life Sciences, Hainan University, Haikou, People’s Republic of China; 2Laboratory of Biopharmaceuticals and Molecular Pharmacology, School of Pharmaceutical Sciences, Hainan University, Haikou, People’s Republic of China; 3One Health Institute, Hainan University, Haikou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yechun Pei, Department of Biosciences, School of Life Sciences, Hainan University, 58 People’s Road, Haikou, Hainan, 570228, People’s Republic of China, Email ycpei@hainanu.edu.cn Dayong Wang, Laboratory of Biopharmaceuticals and Molecular Pharmacology, School of Pharmaceutical Sciences, Hainan University, 58 People’s Road, Haikou, Hainan, 570228, People’s Republic of China, Email wangdy@hainanu.edu.cnBackground: In tropical and subtropical areas, allergens from the dust mite species Blomia tropicalis are common causes of allergic rhinitis and asthma. Blomia tropicalis has two main allergens: Blo t 5 and Blo t 21.Aim: To generate a chimeric virus-like particle containing HBcAg, Blo t 5 and Blo t 21 that can treat allergies caused by Blomia tropicalis.Methods: To produce allergic asthma in mice, prokaryotic expression and purification of Blomia tropicalis allergens rBlo t 5, rBlo t 21, and recombinant fusion allergen rBlo t 5– 21 were utilized in the study. We created a hepatitis B core antigen (HBcAg) and rBlo t 5– 21 fusion prokaryotic expression plasmid. HBcAg-rBlo t 5– 21 was purified after expression and tested by transmission electron microscopy (TEM). Furthermore, the protein HBcAg-rBlo t 5– 21 was employed as a protein vaccination.Results: In allergy-induced mouse model experiments, the fusion allergen rBlo t 5– 21 was more effective than the individual allergens rBlo t 5 and rBlo t 21 at inducing allergy. We found that vaccinating allergic mice with the recombinant fusion protein vaccine HBcAg-rBlo t 5– 21 alleviated allergy symptoms elicited by the rBlo t 5– 21 allergen. Vaccination with HBcAg-rBlo t 5– 21 resulted in a decrease in total serum IgE levels, suppression of anaphylaxis, and reduction of inflammatory cell infiltration into lung tissue as compared to the PBS group.Conclusion: HBcAg-rBlo t 5– 21, a protein vaccine containing both the hepatitis B core antigen and the Blomia tropicalis fusion allergen rBlo t 5– 21, could be a suitable vaccination for preventing allergy disorders caused by Blomia tropicalis.Keywords: Blomia tropicalis, vaccine, hepatitis B core antigen, asthma

Oxidative stress is one of the causes of aging and other diseases. Nrf2 is the main transcription factor regulating antioxidant gene expression. In recent years, it has been found that inhibiting Nrf2 activity or increasing oxidative pressure can replicate premature aging defects, and reactivating Nrf2 activity can reverse nuclear aging defects and restore cell vitality. SIRT6 is one of the few proteins that can regulate life span and aging at the same time. SIRT6 gene deficient mice show degenerative changes similar to human premature aging 3 weeks after birth and eventually die. SIRT6 deficient cells show the characteristics of accelerated aging, while the deletion of SIRT6 in human cells does not directly lead to the damage of genetic material in the nucleus and genomic instability, but significantly reduces the antioxidant capacity of cells and causes the accumulation of intracellular reactive oxygen species and DNA oxidation products. At present, the interaction between Nrf2 and SIRT6 protein and its mechanism are not clear. In this study, yeast two-hybrid and tag pulldown methods were used to analyze the protein interaction domain between Nrf2 and SIRT6. The experiment found that SIRT6 directly interacts with Nrf2, SIRT6 Interacts with the Neh1 and Neh3 domains of Nrf2.