Single-cell RNA-sequencing profiles reveal the developmental landscape of the Manihot esculenta Crantz leaves.

Cassava (Manihot esculenta Crantz) is an important crop with a 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, we created a single-cell transcriptome atlas of cassava leaves. A total of 11,177 high-quality leaf cells were divided into 15 cell clusters. Based on leaf cell marker genes, we identified 3 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 6 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.

Molecular Integrative Analysis of the Inhibitory Effects of Dipeptides on Amyloid ß Peptide 1-42 Polymerization.

The major pathological feature of Alzheimer's disease (AD) is the aggregation of amyloid ß peptide (Aß) in the brain. Inhibition of Aß42 aggregation may prevent the advancement of AD. This study employed molecular dynamics, molecular docking, electron microscopy, circular dichroism, staining of aggregated Aß with ThT, cell viability, and flow cytometry for the detection of reactive oxygen species (ROS) and apoptosis. Aß42 polymerizes into fibrils due to hydrophobic interactions to minimize free energy, adopting a ß-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 Aß42 aggregation the most. The ThT assay and EM revealed that RR reduced Aß42 aggregation, whereas the circular dichroism spectroscopy analysis showed a 62.8% decrease in ß-sheet conformation and a 39.3% increase in random coiling of Aß42 in the presence of RR. RR also significantly reduced the toxicity of Aß42 secreted by SH-SY5Y cells, including cell death, ROS production, and apoptosis. The formation of three hydrophobic regions and polymerization of Aß42 reduced the Gibbs free energy, and RR was the most effective dipeptide at interfering with polymerization.

Aß monomer induces phosphorylation of Tau at Ser-214 through ß2AR-PKA-JNK signaling pathway.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder associated with synaptic dysfunction, pathological accumulation of ß-amyloid peptide 1-42 (Aß1-42 ), and neuronal loss. The self-association of Aß1-42 monomers (Aß-M) into soluble oligomers seems to be crucial for the development of neurotoxicity. Previous publications have shown that Aß oligomers and dimers might play key roles in inducing AD. The role of Aß-M was rarely investigated and still unclear in AD. To understand the effects of Aß-M on neurons and other cell types in the brain could be the key to understand its function. In our study, we found that Aß-M expression slowly induced cell apoptosis within 48 hours after transfection, ß2 adrenergic receptor (ß2AR) interacted with Aß-M in the pull-down and the yeast two-hybrid assays, and Aß-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 ß2AR, G protein-coupled receptor kinase 2 (GRK2), and protein kinase A (PKA) mediated the phosphorylation of Tau and JNK. Aß-M induced phosphorylation of Tau at Ser-214 through both ß2AR-cAMP/PKA-JNK and ß2AR-GRK signaling pathways. Mitogen-activated protein kinase kinase (MEK) mediated the phosphorylation of p70S6K induced by Aß-M.

Stability and Pharmacological Effects of Gene-Recombinant Wild Type and Mutant Human Adrenocorticotropic Hormone.

PURPOSE: 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. METHODS: 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. RESULTS: 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, 3ß-HSD, and SF-1. CONCLUSION: The stability of E5D mutant ACTH is higher than WT ACTH. The pharmacological effects of E5D ACTH is equivalent to those of WT ACTH.

Impaired Mitochondrial Energy Metabolism Regulated by p70S6K: A Putative Pathological Feature in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease. Mitochondrial energy metabolism and p70 ribosomal protein S6 kinase (p70S6K) play significant roles in AD pathology. However, the potential relationship between them is unclear. In this study, bioinformatics methods were initially applied to analyze the transcriptomic data in the CA1 and the primary visual cortex of patients with AD and Aß42-treated SH-SY5Y cells. By applying secreted Aß42 and p70S6K gene silencing in cells, we explored disorders in mitochondrial function and the regulatory roles of p70S6K by flow cytometry, laser scanning confocal microscopy, high-performance liquid chromatography, Western blotting, and quantitative reverse transcription PCR. The study reveals that impaired mitochondrial energy metabolism is a potential pathological feature of AD and that p70S6K gene silencing reversed most of the changes induced by Aß42, such as the activities of the electron transport chain complexes I and III, as well as ATP synthase, ATP production, generation of reactive oxygen species, mitochondrial membrane potential, and phosphorylation of AMPK, PINK1, and Parkin, all of which are required for mitochondria to function properly in the cell.

A Molecular Integrative Study on the Inhibitory Effects of WRR and ERW on Amyloid ß Peptide (1-42) Polymerization and Cell Toxicity.

Alzheimer's disease (AD) is a neurodegenerative disease and the main pathological characteristic of AD is the deposition of Aß42 in the brain. Inhibition of Aß42 polymerization is one of the important research directions. Due to the pathological complexity of Alzheimer's disease, studies on Aß42 polymerization inhibitors have not made significant progress worldwide. Using an independently constructed structure database of oligopeptides, in this study, molecular docking, umbrella sampling analysis of free energy, ThT fluorescence detection of Aß42 polymerization, transmission electron microscopy, and flow cytometry detection of reactive oxygen species (ROS) and apoptosis were performed to screen tripeptides and pentapeptides that inhibit polymerization. It was found that two tripeptides, i.e., WRR and ERW, bind stably to the core of Aß42 polymerization in the molecular dynamics analysis, and they significantly inhibited the aggregation of Aß42 and reduced their cell toxicity in vitro.

Molecular integrative study on interaction domains of nuclear factor erythroid 2-related factor 2 with sirtuin 6.

Oxidative stress is one of the elements causing aging and related diseases. Inhibiting Nrf2 activity or increasing oxidative pressure can replicate the deficits of premature aging. SIRT6 is one of the few proteins that can regulate both life span and aging. Deletion of SIRT6 in human cells impairs the antioxidant capacity of cells, which results in the accumulation of intracellular reactive oxygen species and DNA oxidation products. Characterization of the binding of Nrf2 with SIRT6 is critical for understanding the modulation of Nrf2-correlated cell activities by SIRT6. The yeast two-hybrid experiments showed that the binding of Nrf2 with SIRT6 is mediated by Neh1 and Neh3 domains. The elimination of the Neh1 and Neh3 domains decreased the binding stability and free energy, according to the molecular dynamic analysis. The roles of theses domains in mediating the binding were confirmed by co-immunoprecipitation. In cells transfected with the small interfering RNA (siRNA) targeting the Nrf2 Neh1 domain and plasmids overexpressing domain-mutant Nrf2, it was discovered that Nrf2 lost its activity to stimulate the transcription of antioxidant genes in the absence of Neh1 and Neh3 domains.

Studies on HBcAg-rBlo t 5-21 Fusion Protein Vaccine That Alleviates Blomia tropicalis Airway Inflammation.

Background: 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.

Molecular dynamic and pharmacological studies on protein-engineered hirudin variants of Hirudinaria manillensis and Hirudo medicinalis.

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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 Ki and IC50 values. In the in vivo experiments, mutations at C-termini of HV1 and HM2 significantly changed partial thromboplastin time, prothrombin and thrombin time CONCLUSION AND IMPLICATIONS: The study indicated that the anticoagulant effects of gene-engineered HM2 are stronger than gene-engineered HV1 and HM2-E60D-I62D has the strongest effects and could be an antithrombotic with better therapeutic effects.

Role of Dietary Gluten in Development of Celiac Disease and Type I Diabetes: Management Beyond Gluten-Free Diet.

Gluten triggers Celiac Disease (CD) and type I diabetes in genetically predisposed population of human leukocyte antigen DQ2/DQ8+ and associates with disorders such as schizophrenia and autism. Application of a strict gluten-free diet is the only well-established treatment for patients with CD, whereas the treatment for patients with celiac type I diabetes may be depend on the timing and frequency of the diet. The application of a gluten-free diet in patients with CD may contribute to the development of metabolic syndrome and nonalcoholic fatty liver disease and may also lead to a high glycemic index, low fiber diet and micronutrient deficiencies. The alteration of copper bioavailability (deficient, excess or aberrant coordination) may contribute to the onset and progress of related pathologies. Therefore, nutrient intake of patients on a gluten-free diet should be the focus of future researches. Other gluten-based therapies have been rising with interest such as enzymatic pretreatment of gluten, oral enzyme supplements to digest dietary gluten, gluten removal by breeding wheat varieties with reduced or deleted gluten toxicity, the development of polymeric binders to suppress gluten induced pathology.

Sustained Stimulation of ß2AR Inhibits Insulin Signaling in H9C2 Cardiomyoblast Cells Through the PKA-Dependent Signaling Pathway.

INTRODUCTION: This study aimed to investigate the role of ß2 adrenergic receptor (ß2AR) in insulin signaling transduction in H9C2 cardiomyoblast cells to understand the formation of the ß2AR-insulin receptor (IR) protein complex and its role in insulin-induced Glut4 expression. METHODS: H9C2 cells were treated with various protein inhibitors (CGP, ß1AR inhibitor CGP20712; ICI, ß2AR inhibitor ICI 118,551; PKI, PKA inhibitor myristoylated PKI; PD 0325901, MEK inhibitor; SP600125, JNK inhibitor) with or without insulin or isoproterenol (ISO) before RNA-sequencing (RNA-Seq) and quantitative-PCR (Q-PCR). Yeast two-hybrid, co-immunoprecipitation and His-tag pull-down assay were carried out to investigate the formation of the ß2AR-IR protein complex. The intracellular concentrations of cAMP in H9C2 cells were tested by high performance liquid chromatography (HPLC) and the phosphorylation of JNK was tested by Western blot. RESULTS: Gene Ontology (GO) analysis revealed that the most significantly enriched processes in the domain of molecular function (MF) were catalytic activity and binding, whereas in the domain of biological processes (BP) were metabolic process and cellular process. Furthermore, the enriched processes in the domain of cellular components (CC) were cell and cell parts. The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the most significant pathways that have been altered included the PI3K-Akt and MAPK signaling pathways. Q-PCR, which was performed to verify the gene expression levels exhibited consistent results. In evaluating the signaling pathways, the sustained stimulation of ß2AR by ISO inhibited insulin signalling, and the effect was primarily through the cAMP-PKA-JNK pathway and MEK/JNK signaling pathway. Yeast two-hybrid, co-immunoprecipitation and His-tag pull-down assay revealed that ß2AR, IR, insulin receptor substrate 1 (IRS1), Grb2-associated binding protein 1 (GAB1) and Grb2 existed in the same protein complex. CONCLUSION: The sustained stimulation of ß2AR might inhibit insulin signaling transduction through the cAMP-PKA-JNK and MEK/JNK pathways in H9C2 cells.

Role of human Keap1 S53 and S293 residues in modulating the binding of Keap1 to Nrf2.

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.

Profiling of Differential Expression of Genes in Mice Carrying Both Mutant Presenilin 1 and Amyloid Precursor Protein Transgenes with or without Knockout of B2 Adrenergic Receptor Gene.

Alzheimer's disease (AD) is a lifelong progressive neurodegenerativa disease related with accumulation of amyloid ß peptide (Aß) 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 ß2AR gene knock-out (ß2AR-KO) is beneficial in senile AD animals. Consistently, an epidemiological study lasted for two decades showed that the sole usage of ß 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 ß2AR-KO AD mice had better learning and memory, genomic effects of ß2AR-KO in the double transgenic AD mice were investigated. In the analysis, major genomic significance of ß2AR-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 ß peptide, the hallmark of AD.

Transcriptomic analysis of gene expression in mice treated with troxerutin.

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).

Induced Regulatory T Cells Superimpose Their Suppressive Capacity with Effector T Cells in Lymph Nodes via Antigen-Specific S1p1-Dependent Egress Blockage.

Regulatory T cells (Tregs) restrict overexuberant lymphocyte activation. While close proximity between Tregs and their suppression targets is important for optimal inhibition, and literature indicates that draining lymph nodes (LNs) 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 LN, downregulate 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 LNs 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.

Fel d 1-airway inflammation prevention and treatment by co-immunization vaccine via induction of CD4+CD25-Foxp3+ Treg cells.

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-ß 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.