Mechanism of active acetylcholinesterase inhibition by organic sulfanes in garlic: Non-covalent binding and covalent modifications.

Numerous secondary metabolites in medicinal food homology plants such as Allium inhibit the activity of acetylcholinesterase (AChE), but the current understanding of the inhibition mechanism is limited. In this study, we employed ultrafiltration, spectroscopic, molecular docking, and matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) techniques to investigate the inhibition mechanism of AChE by garlic organic sulfanes, including diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS). The results of UV-spectrophotometry and ultrafiltration experiments showed the inhibition of AChE activity by DAS and DADS was reversible (competitive inhibition), but inhibition by DATS was irreversible. Molecular fluorescence and molecular docking indicated DAS and DADS changed the positions of key amino acids inside the catalytic cavity through hydrophobic interactions with AChE. By using MALDI-TOF-MS/MS, we found DATS irreversibly inhibited AChE activity by opening disulfide-bond switching of disulfide bond 1 (Cys-69 and Cys-96) and disulfide bond 2 (Cys-257 and Cys-272) in AChE, as well as by covalently modifying Cys-272 in disulfide bond 2 to generate AChE-SSA derivatives (strengthened switch). This study provides a basis for further exploration of natural AChE inhibitors using organic active substances in garlic and presents a hypothesis of U-shaped spring force arm effect based on the disulfide bond-switching reaction of DATS that can be used to evaluate the stability of disulfide bonds in proteins.

Effect of heat-treating methods on components, instrumental evaluation of color and taste, and antioxidant properties of sea buckthorn pulp flavonoids.

Sea buckthorn has strong physiological activities, which might be attributed to its flavonoid compounds. The heat treatment could speed up the transformation of the flavonoids, so it would affect the properties of the flavonoids. To explore the effect of the heat-treating methods on the sea buckthorn pulp flavonoids, the flavonoid compositions, color and taste properties, and antioxidant activity, prepared by three heating methods, were investigated. The results showed that the extractable sea buckthorn flavonoids rose after heat treatment; the high temperature with high-pressure treatment had the largest yield (3.66 ± 0.21 mg/ml). A total of 16 flavonoid monomers were identified in the sea buckthorn, the content of isorhamnetin-3-O-rutinoside was the highest in the pulp (589.34 ± 4.72 µg/ml), the concentrations of eight monomers increased after heat-treating, and the treating methods could significantly affect the contents of flavonoid monomers (p < 0.05). The difference in color was positively correlated to the heat-treating time and temperature (p < 0.01); the ΔE value in high pressure with high temperature treatment was the largest (10.59). The heat treatment enhanced bitterness and acidity, while it greatly reduced the saltiness, umami, and sweetness (p < 0.01). The stronger antioxidant capacity of the total flavonoids was accompanied by the higher temperature and longer time of heat treatment. The antioxidant capacity was closely correlated to the contents of flavonoid monomers. Therefore, the heat treatment boosted the content of the flavonoids, enhanced the taste, and improved the antioxidant properties. PRACTICAL APPLICATION: This study can provide suitable a method for improving the extracting efficiency, improving properties and biological activities of sea buckthorn pulp flavonoids, also provide directions for predicting the antioxidant activity by the flavonoid monomer contents.

Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment.

COVID-19, caused by SARS-CoV-2, is a virulent pneumonia, with >4,000,000 confirmed cases worldwide and >290,000 deaths as of May 15, 2020. It is critical that vaccines and therapeutics be developed very rapidly. Mice, the ideal animal for assessing such interventions, are resistant to SARS-CoV-2. Here, we overcome this difficulty by exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2). Ad5-hACE2-sensitized mice developed pneumonia characterized by weight loss, severe pulmonary pathology, and high-titer virus replication in lungs. Type I interferon, T cells, and, most importantly, signal transducer and activator of transcription 1 (STAT1) are critical for virus clearance and disease resolution in these mice. Ad5-hACE2-transduced mice enabled rapid assessments of a vaccine candidate, of human convalescent plasma, and of two antiviral therapies (poly I:C and remdesivir). In summary, we describe a murine model of broad and immediate utility to investigate COVID-19 pathogenesis and to evaluate new therapies and vaccines.

The murine IgH locus contains a distinct DNA sequence motif for the chromatin regulatory factor CTCF.

Antigen receptor assembly in lymphocytes involves stringently-regulated coordination of specific DNA rearrangement events across several large chromosomal domains. Previous studies indicate that transcription factors such as paired box 5 (PAX5), Yin Yang 1 (YY1), and CCCTC-binding factor (CTCF) play a role in regulating the accessibility of the antigen receptor loci to the V(D)J recombinase, which is required for these rearrangements. To gain clues about the role of CTCF binding at the murine immunoglobulin heavy chain (IgH) locus, we utilized a computational approach that identified 144 putative CTCF-binding sites within this locus. We found that these CTCF sites share a consensus motif distinct from other CTCF sites in the mouse genome. Additionally, we could divide these CTCF sites into three categories: intergenic sites remote from any coding element, upstream sites present within 8 kb of the VH-leader exon, and recombination signal sequence (RSS)-associated sites characteristically located at a fixed distance (∼18 bp) downstream of the RSS. We noted that the intergenic and upstream sites are located in the distal portion of the VH locus, whereas the RSS-associated sites are located in the DH-proximal region. Computational analysis indicated that the prevalence of CTCF-binding sites at the IgH locus is evolutionarily conserved. In all species analyzed, these sites exhibit a striking strand-orientation bias, with >98% of the murine sites being present in one orientation with respect to VH gene transcription. Electrophoretic mobility shift and enhancer-blocking assays and ChIP-chip analysis confirmed CTCF binding to these sites both in vitro and in vivo.

[Clinical trial of treatment of laryngeal cancer by recombinant adenovirus p53 combined with chemotherapy].

OBJECTIVE: To evaluate the effectiveness of recombinant adenovirus p53 injection (rAd2p53) combined with cisplatin plus 5-fluorouracil regimen in treating laryngeal carcinoma. METHOD: Tumour animal models were established in the back of mice with Hep-2 cell line. Recombinant adenovirus p53 injection (rAd2p53) were transduced to tumor-bearing mouse by direct intratumoral injection combine with cisplatin plus 5-fluorouracil ivgtt. The control group 1 was given recombinant adenovirus p53 injection (rAd2p53) simplex. The control group 2 was administered with cisplatin plus 5-fluorouracil ivgtt simplex. Then compare the diameters of pro-treatment with that of post-treatment and test group with controls. RESULT: Tumor growth was significantly inhibited following combined rAd2p53 gene therapy with cisplatin plus 5-fluorouracil chemotherapy compared to single rAd2p53 gene therapy and cisplatin plus 5-fluorouracil chemotherapy controls. CONCLUSION: Local injection of rAd2p53 combined with cisplatin plus 5-fluorouracil chemotherapy is a promising treatment to laryngeal cancer.