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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.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder associated with synaptic dysfunction, pathological accumulation of β-amyloid peptide 1-42 (Aβ1-42 ), and neuronal loss. The self-association of Aβ1-42 monomers (Aβ-M) into soluble oligomers seems to be crucial for the development of neurotoxicity. Previous publications have shown that Aβ oligomers and dimers might play key roles in inducing AD. The role of Aβ-M was rarely investigated and still unclear in AD. To understand the effects of Aβ-M on neurons and other cell types in the brain could be the key to understand its function. In our study, we found that Aβ-M expression slowly induced cell apoptosis within 48 hours after transfection, β2 adrenergic receptor (β2AR) interacted with Aβ-M in the pull-down and the yeast two-hybrid assays, and Aβ-M played a major role in inducing phosphorylation of Tau at Ser-214, c-Jun N-terminal kinase (JNK) at Thr-183/Tyr-185, p70 ribosomal protein S6 kinase (p70S6K) at Thr-389. We also discovered that β2AR, G protein-coupled receptor kinase 2 (GRK2), and protein kinase A (PKA) mediated the phosphorylation of Tau and JNK. Aβ-M induced phosphorylation of Tau at Ser-214 through both β2AR-cAMP/PKA-JNK and β2AR-GRK signaling pathways. Mitogen-activated protein kinase kinase (MEK) mediated the phosphorylation of p70S6K induced by Aβ-M.

Alzheimer’s disease (AD) is a lifelong progressive neurodegenerativa disease related with accumulation of amyloid β peptide (Aβ) produced by processing of amyloid precursor protein (APP) in the brain. In spite of several-decades effort on AD, there is still no medicine used to intervene with its pathological processes. Our previous studies made in transgenic animal models harboring familial AD genes of mutant presenilin 1 and amyloid precursor protein (APP) showed that β(2)AR gene knock-out (β(2)AR-KO) is beneficial in senile AD animals. Consistently, an epidemiological study lasted for two decades showed that the sole usage of β blockers as antihypertensive medicines is associated with fewer brain lesions and less brain shrinkage seen in senile AD patients. In order to understand why senile β(2)AR-KO AD mice had better learning and memory, genomic effects of β(2)AR-KO in the double transgenic AD mice were investigated. In the analysis, major genomic significance of β(2)AR-KO was directed to influence protein-processing and presentation involving membrane structure and MHC class I and II protein complex, and lysosome and hydrolase activity for protein degradation, which are critical for accumulation of amyloid β peptide, the hallmark of AD.

Keap1 is deemed as a suppressor of Nrf2 in cytoplasm by sequestrating Nrf2 to proteolysis as an adapter of the Cul3-Rbx1 E3 ubiquitin ligase complex. In the study, it was proposed that post-translational modification might affect the interaction between Nrf2 and Keap1, and the profiles of the phosphorylation of amino acid residues of Keap1 and its effects on the binding of Keap1 to Nrf2 was investigated. A mass spectrometry analysis revealed that S53 and S293 were phosphorylated upon an oxidative stress. Using Keap1 proteins with amino acid residues mutated to glutamate to simulate the introduction of a negative charge by phosphorylation, it was found that a potential phosphorylation of S53 affected Keap1-Nrf2 binding in the pull-down assay, and induced nuclear translocation of Nrf2 in the electrophoretic mobility shift assay. Sequence homology analysis showed that S53 was highly conserved. Structural modeling around BTB domain of wild type and S53E-mutant Keap1 showed that the negative charge introduced by S53E mutation generates a salt bridge between E53 and ionized guanidine group of Arg50. Real-time qRT-PCR for transcription levels of antioxidant genes that are modulated by Nrf2 further proved the effects of the potential phosphorylation of S53 under an oxidative stress condition. In summary, S53 is a potential phosphorylation site of Keap1, and the phosphorylation could enhance the antioxidative capacity of cells in response to an oxidative stress.

Alzheimer's disease (AD) is a lifelong progressive neurodegenerativa disease related with accumulation of amyloid β peptide (Aβ) produced by processing of amyloid precursor protein (APP) in the brain. In spite of several-decades effort on AD, there is still no medicine used to intervene with its pathological processes. Our previous studies made in transgenic animal models harboring familial AD genes of mutant presenilin 1 and amyloid precursor protein (APP) showed that β2AR gene knock-out (β2AR-KO) is beneficial in senile AD animals. Consistently, an epidemiological study lasted for two decades showed that the sole usage of β blockers as antihypertensive medicines is associated with fewer brain lesions and less brain shrinkage seen in senile AD patients. In order to understand why senile β2AR-KO AD mice had better learning and memory, genomic effects of β2AR-KO in the double transgenic AD mice were investigated. In the analysis, major genomic significance of β2AR-KO was directed to influence protein-processing and presentation involving membrane structure and MHC class I and II protein complex, and lysosome and hydrolase activity for protein degradation, which are critical for accumulation of amyloid β peptide, the hallmark of AD.

Troxerutin, a semi-synthetic derivative of the natural bioflavanoid rutin, has been reported to possess many beneficial effects in human bodies, such as vasoprotection, immune support, anti-inflammation and anti-aging. However, the effects of troxerutin on genome-wide transcription in blood cells are still unknown. In order to find out effects of troxerutin on gene transcription, a high-throughput RNA sequencing was employed to analysis differential gene expression in blood cells consisting of leucocytes, erythrocytes and platelets isolated from the mice received subcutaneous injection of troxerutin. Transcriptome analysis demonstrated that the expression of only fifteen genes was significantly changed by the treatment with troxerutin, among which 5 genes were up-regulated and 10 genes were down-regulated. Bioinformatic analysis of the fifteen differentially expressed genes was made by utilizing the Gene Ontology (GO), and the differential expression induced by troxerutin was further evaluated by real-time quantitative PCR (Q-PCR).

Regulatory T cells (Tregs) restrict over-exuberant lymphocyte activation. While close proximity between Tregs and their suppression targets is important for optimal inhibition, and literature indicates that draining lymph nodes may serve as a prime location for the suppression, signaling details orchestrating this event are not fully characterized. Using a protocol to enable peripheral generation of inducible antigen-specific Tregs (asTregs) to control allergen-induced asthma, we have identified an antigen-specific mechanism that locks asTregs within hilar LNs which in turn suppresses airway inflammation. The suppressive asTregs, upon antigen stimulation in the lymph node, downregulate S1p1 (Sphingosine-1-phosphate receptor 1) egress receptor expression. These asTregs in turn mediate the downregulation of the same receptor on incoming effector T cells. Therefore, asTregs and effector T cells are locked in these draining lymph nodes for prolonged interactions. Disruption of individual steps of this retention sequence abolishes the inflammation controlled by asTregs. Collectively, this study identifies a new requirement of spatial congregation with their suppression targets essential for asTreg functions, and suggests therapeutic programs via Treg traffic control.

Pet allergens are major causes for asthma and allergic rhinitis. Fel d 1 protein, a key pet allergen from domestic cat, can sensitize host and trigger asthma attack. In this study, we report that co-immunization with recombinant Fel d 1 protein (rFel d 1) plus plasmid DNA that contains Fe1 d 1 gene was effective in preventing and treating the natural Fel d 1 (nFel d 1) induced allergic airway inflammation in mice. A population of T regulatory cells (iTreg) exhibiting a CD4+CD25-Foxp3+ phenotype and expressing IL-10 and TGF-β was induced by this co-immunization strategy. Furthermore, after adoptive transfers of the iTreg cells, mice that were pre-sensitized and challenged with nFel d 1 exhibited less signs of allergic inflammation, AHR and a reduced allergic immune response. These data indicate that co-immunization with DNA and protein mixture vaccine may be an effective treatment for cat allergy.

Adrenocorticotropic hormone (ACTH) is the only medicine for treating infantile spasms, however, it is catabolized rapidly. In order to make an ACTH derivative with prolonged effects, we prepared genetically engineered wild type (WT) and mutant ACTH candidates based on protease database analysis, and compared their stability and pharmacological effects. For analysis of stability, serum concentration of WT and mutant ACTH candidates were tested at different time after intravenous injection, and elimination curves were calculated to compare pharmacokinetic properties of WT and E5D-mutant ACTH. For comparison of their pharmacological effects, levels of glucocorticoids (GC) in the blood serum and secreted from cultured Y1 mouse adrenal cells were tested, and their effects on the signaling pathway mediating the expression of genes critical for GC synthesis were analyzed. The effects of ACTHs on transcription levels of the genes involved in GC synthesis were tested by qPCR. The blood concentration of E5D ACTH is higher than the WT after injection, and E5D mutation increased the t1/2 and AUC of ACTH. Pharmacological experiments showed that the effects of E5D and Y2S mutant ACTH on the production of GC and the critical signal transduction were equivalent to those of WT. WT, E5D and Y2S ACTH also have similar effects on the transcriptional levels of the genes for GC synthesis, including STAR, P450-scc, 3β-HSD, and SF-1. The stability of E5D mutant ACTH is higher than WT ACTH. The pharmacological effects of E5D ACTH is equivalent to those of WT ACTH.