Enzymatic ligation of an antibody and arginine 9 peptide for efficient and cell-specific siRNA delivery

A fusion protein comprising an antibody and a cationic peptide, such as arginine-9 (R9), is a candidate molecule for efficient and cell-specific delivery of siRNA into cells in order to reduce the side effects of nucleic acid drugs. However, their expression in bacterial hosts, required for their development, often fails, impeding research progress. In this study, we separately prepared anti-EGFR nanobodies with the K-tag sequence MRHKGS at the C-terminus and R9 with the Q-tag sequence LLQG at the N-terminus, and enzymatically ligated them in vitro by microbial transglutaminase to generate Nanobody-R9, which is not expressed as a fused protein in E. coli. Nanobody-R9 was synthesized at a maximum binding efficiency of 85.1%, without changing the binding affinity of the nanobody for the antigen. Nanobody-R9 successfully delivered siRNA into the cells, and the cellular influx of siRNA increased with increase in the ratio of Nanobody-R9 to siRNA. We further demonstrated that the Nanobody-R9–siRNA complex, at a 30:1 ratio, induced an approximately 58.6% reduction in the amount of target protein due to RNAi in mRNA compared to lipofectamine.

A novel yeast-based high-throughput method for the identification of protein palmitoylation inhibitors

Protein S-acylation or palmitoylation is a widespread post-translational modification that consists of the addition of a lipid molecule to cysteine residues of proteins through a thioester bond. Palmitoylation and palmitoyltransferases (PATs) have been linked to several types of cancers, diseases of the central nervous system and many infectious diseases where pathogens use the host cell machinery to palmitoylate their effectors. Despite the central importance of palmitoylation in cell physiology and disease, progress in the field has been hampered by the lack of potent-specific inhibitors of palmitoylation in general, and of individual PATs in particular. Herein, we present a yeast-based method for the high-throughput identification of small molecules that inhibit protein palmitoylation. The system is based on a reporter gene that responds to the acylation status of a palmitoylation substrate fused to a transcription factor. The method can be applied to heterologous PATs such as human DHHC20, mouse DHHC21 and also a PAT from the parasite Giardia lamblia . As a proof-of-principle, we screened for molecules that inhibit the palmitoylation of Yck2, a substrate of the yeast PAT Akr1. We tested 3200 compounds and were able to identify a candidate molecule, supporting the validity of our method.

Synthesis and Acivity of Chiral Amino Alcohols and Amides as New Cholinesterase Inhibitors for Alzheimer's Disease

A series of biological active compounds 1–14 have been synthesized and used as potential inhibitors for AChE and BuChE. Potential inhibitor efficacy of these molecules to the target enzymes have been searched in vitro and theoretical by dock and molecular dynamic calculations. The results show that chiral amino alcohol compounds 6, 7 and 9 exhibited good value for medication scores. Among the tested compounds the best inhibition activities have been obtained with compounds 6 for AChE and BuChE by 87.68 and 92.46 % values, respectively at 50µg/mL concentration. The anticipated value of 6 also justified superb correlation with invitro statistics and it could be taken into consideration as drug candidate molecule for designing of novel drug. Potential inhibitory outcomes of those molecules on the right track proteins were investigated the use of Docking and Molecular Dynamics calculations. Dock score evaluation and Lipinski parameters have been proven those ligands are ability inhibitors against applicable enzymes. Our findings endorse that related compounds can be applied as a capacity supply of anti-alzheimer active molecules for designing novel products.

Hydrogen Sulfide Metabolite, Sodium Thiosulfate: Clinical Applications and Underlying Molecular Mechanisms

Thiosulfate in the form of sodium thiosulfate (STS) is a major oxidation product of hydrogen sulfide (H2S), an endogenous signaling molecule and the third member of the gasotransmitter family. STS is currently used in the clinical treatment of acute cyanide poisoning, cisplatin toxicities in cancer therapy, and calciphylaxis in dialysis patients. Burgeoning evidence show that STS has antioxidant and anti-inflammatory properties, making it a potential therapeutic candidate molecule that can target multiple molecular pathways in various diseases and drug-induced toxicities. This review discusses the biochemical and molecular pathways in the generation of STS from H2S, its clinical usefulness, and potential clinical applications, as well as the molecular mechanisms underlying these clinical applications and a future perspective in kidney transplantation.

In Vitro Evaluation of Immunogenicity of Recombinant OMP25 Protein Obtained from Endemic Brucella abortus Biovar 3 as Vaccine Candidate Molecule Against Animal Brucellosis

Background: Brucellosis is a zoonotic disease that causes serious economic losses due to factors such as miscarriages and decreased milk yield in animals. Existing live vaccines have some disadvantages, so effective vaccines need to be developed with new technological approaches. Objectives: The primary objectives of this study were the expression and purification of recombinant Omp25 fusion protein from B. abortus and the evaluation of the effect of the Omp25 protein on cell viability and inflammatory response. Methods: The omp25 gene region was amplified by a polymerase chain reaction and cloned into a Pet102/D-TOPO expression vector. The protein expression was carried out using the procaryotic expression system. The recombinant Omp25 protein was purified with affinity chromatography followed by GPC (Gel Permeation Chromatography). The MTS assay and cytokine-release measurements were carried out to evaluate cell viability and inflammatory response, respectively Results: It was determined that doses of the recombinant Omp25 protein doses greater than 0.1 μg/mL are toxic to RAW cells. Doses of 1 µg/mL and lower significantly increased inflammation due to nitric oxide (NO) levels. ELISA results show that IFN-γ was produced in stimulated RAW 264.7 cells at a dose that did not affect the viability (0.05 µg/mL). However, IL-12, which is known to have a dual role in the activation of macrophages, did not show a statistically significant difference at the same dose. Conclusion: Studies of cell viability and Th1‐related cytokine release suggest that Omp25 protein is a promising candidate molecule for vaccine development.

Enzymatic Ligation of Antibody and Cell-Penetrating Peptide for Efficient and Cell-Specific siRNA Delivery

A fusion protein comprising an antibody and a cell-penetrating peptide is a candidate molecule capable of efficient and cell-specific delivery of siRNA into cells in order to reduce the side effects of nucleic acid drugs. However, their expression in bacterial hosts, required for their development, often fails, impeding research progress. In this study, we separately prepared anti-EGFR nanobodies with the K-tag sequence MRHKGS at the C-terminus and arginine-9 (R9) with the Q-tag sequence LLQGS at the N-terminus, and enzymatically ligated them in vitro by microbial transglutaminase to generate Nanobody-R9, which is not expressed as a fused protein in E. coli. Nanobody-R9 exhibited a maximum reaction efficiency of 85.1%, without changing the properties of the nanobody or R9. Nanobody-R9 successfully delivered siRNA into the cells, and the cellular influx of siRNA increased with increase in the ratio of Nanobody-R9 to siRNA. We further demonstrated that the Nanobody-R9–siRNA complex, at a 30:1 ratio, induced RNAi of target mRNA with approximately 52% efficiency compared to lipofectamine.

Intermittent fasting enhances long-term memory consolidation, adult hippocampal neurogenesis, and expression of longevity gene Klotho

Daily calorie restriction (CR) and intermittent fasting (IF) enhance longevity and cognition but the effects and mechanisms that differentiate these two paradigms are unknown. We examined whether IF in the form of every-other-day feeding enhances cognition and adult hippocampal neurogenesis (AHN) when compared to a matched 10% daily CR intake and ad libitum conditions. After 3 months under IF, female C57BL6 mice exhibited improved long-term memory retention. IF increased the number of BrdU-labeled cells and neuroblasts in the hippocampus, and microarray analysis revealed that the longevity gene Klotho (Kl) was upregulated in the hippocampus by IF only. Furthermore, we found that downregulating Kl in human hippocampal progenitor cells led to decreased neurogenesis, whereas Kl overexpression increased neurogenesis. Finally, histological analysis of Kl knockout mice brains revealed that Kl is required for AHN, particularly in the dorsal hippocampus. These data suggest that IF is superior to 10% CR in enhancing memory and identifies Kl as a novel candidate molecule that regulates the effects of IF on cognition likely via AHN enhancement.

Formation of fibrin at sights of conceptus adhesion in the ewe

In ruminants, various molecules are involved in regulating conceptus attachment and adhesion; however, molecules that maintain the conceptus adhesion have not been well characterized. We hypothesized that conceptus must produce a molecule(s), yet uncharacterized or overlooked, which maintain conceptus adhesion to the uterine epithelium. In this study, we aimed to identify new candidate(s) in conceptus secretory proteins responsible for maintaining conceptus adhesion in sheep. We performed RNA-sequence analysis with ovine conceptuses, followed by endometria obtained from pregnant animals on day 15 (P15: pre-attachment), 17 (P17: right after attachment), and 21 (P21: post-attachment; adhesion) and iTRAQ analysis of uterine flushing on P15 and P17. To identify the proteins secreted from conceptuses, we cross-referenced the transcriptome and proteome data. These analyses identified 16 and 26 proteins as conceptus secretory proteins on P15 and P17, respectively. Gene ontology analysis revealed that the conceptus secretory proteins were enriched in those categorized to fibrinolysis and coagulation. RT-qPCR analysis verified that the expression levels of transcripts in conceptuses encoding coagulation factors, fibrinogen subunits, and fibrinolysis factors were significantly higher on P21 than on P15 or P17, which were supported by those through in situ hybridization, western blotting and immunohistochemistry. Histology analysis confirmed that fibrin protein was present at the conceptus adhesion region on P21. These results suggest that in addition to the numerous adhesion molecules so far characterized, fibrin is a new candidate molecule for maintaining conceptus adhesion for pregnancy continuation in ruminants.

Nutritional Composition and Mineral Elements Content Analyses of Lonicera fulvotomentosa Hsu et S. C. Cheng Grown in China

Lonicera fulvotomentosa Hsu et S. C. Cheng (L. fulvotomentosa), a vine shrub found in Southwestern China, is used for treating epidemic fever and infectious diseases, such as SARS and Avian Influenza. Here, we investigated the chemical composition and nutritional content of dried flowers of L. fulvotomentosa grown in yellow loam and Karst landform soil in Guizhou, China. The moisture content in all samples varied from 3.25 to 3.63%, lipids from 7.76 to 9.93%, fiber from 6.93 to 7.34%, ashes from 12.32 to 12.76%, crude protein from 7.85 to 8.53%, and carbohydrates from 56.21 to 59.77%. Using inductively coupled plasma-mass spectroscopy (ICP-MS), the predominant mineral elements in the dried flowers were found to be calcium (297.34-351.26 mg/kg), potassium (132.56-140.37 mg/kg), iron (37.77–41.25 mg/kg), and magnesium (9.47–11.36 mg/kg). Also, HPLC identified flavonoids (kaempferol, rutin, quercetin, luteolin, and apigenin) and phenolic acids (caffeic acid, gallic acid, and chlorogenic acid). Thus, the chemical composition of L. fulvotomentosa was similar to that of Lonicera japonica Thunb. (L. japonica). Thus, it could be used as an alternative to L. japonica. Our results showed that the dried flower of L. fulvotomentosa had an extremely high content of chlorogenic acids and caffeic acid, which could be developed as a candidate molecule as HIV inhibitors.