ABSTRACT
An analytical method for 10 mycotoxins in Hippophae Fructus medicinal and edible products was established in this study, and the contamination of their mycotoxins was analyzed. First of all, the mixed reference solution of ten mycotoxins such as aflatoxin, ochratoxin, zearalenone, and dexoynivalenol was selected as the control, and the Hippophae Fructus medicinal and edible products were prepared. Secondly, based on the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) technology, 10 mycotoxins in Hippophae Fructus medicinal and edible products were quantitatively investigated and their content was determined. Finally, the contamination of mycotoxins was analyzed and evaluated. The optimal analysis conditions were determined, and the methodological inspection results showed that the 10 mycotoxins established a good linear relationship(r>0.99). The method had good repeatability, test sample specificity, stability, and instrument precision. The average recovery rates of 10 mycotoxins in Hippophae Fructus medicinal products, edible solids, and edible liquids were 90.31%-109.4%, 87.86%-107.8%, and 85.61%-109.1%, respectively. Relative standard deviation(RSD) values were 0.22%-10%, 0.75%-13%, and 0.84%-8.5%, repsectively. Based on UPLC-MS/MS technology, the simultaneous determination method for the limits of 10 mycotoxins established in this study has fast detection speed, less matrix interference, high sensitivity, and accurate results, which is suitable for the limit examination of 10 mycoto-xins in Hippophae Fructus medicinal and edible products.
Subject(s)
Mycotoxins/analysis , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Hippophae , Limit of Detection , Chromatography, High Pressure Liquid/methodsABSTRACT
Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness. One-Sentence SummaryWe designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.
ABSTRACT
Antibodies are widely used in biology and medicine, and there has been considerable interest in multivalent antibody formats to increase binding avidity and enhance signaling pathway agonism. However, there are currently no general approaches for forming precisely oriented antibody assemblies with controlled valency. We describe the computational design of two-component nanocages that overcome this limitation by uniting form and function. One structural component is any antibody or Fc fusion and the second is a designed Fc-binding homo-oligomer that drives nanocage assembly. Structures of 8 antibody nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage match the corresponding computational models. Antibody nanocages targeting cell-surface receptors enhance signaling compared to free antibodies or Fc-fusions in DR5-mediated apoptosis, Tie2-mediated angiogenesis, CD40 activation, and T cell proliferation; nanocage assembly also increases SARS-CoV-2 pseudovirus neutralization by -SARS-CoV-2 monoclonal antibodies and Fc-ACE2 fusion proteins. We anticipate that the ability to assemble arbitrary antibodies without need for covalent modification into highly ordered assemblies with different geometries and valencies will have broad impact in biology and medicine.
ABSTRACT
To test the hypothesis that AMP-activated protein kinase (AMPK) is possibly the downstream signaling molecule of certain subtypes of adrenergic receptor (AR) in the heart, we evaluated AMPK activation mediated by ARs in H9C2 cells, a rat cardiac source cell line, and rat hearts. The AMPK-alpha subunit and the phosphorylation level of Thr(172)-AMPK-alpha subunit were subjected to Western blot analysis. Osmotic minipumps filled with norepinephrine (NE), phenylephrine (PE) or vehicle [0.01% (W/V) vitamin C solution] were implanted into male Sprague-Dawley rats subcutaneously. The pumps delivered NE or PE continuously at the rate of 0.2 mg/kg per hour. After 7-day infusion, the activity of AMPK was examined following immunoprecipitation with anti-AMPK-alpha antibody. At the cellular level, we found that NE elevated AMPK phosphorylation level in a dose- and time-dependent manner, with the maximal effect at 10 micromol/L NE after 10-minute treatment. This effect was insensitive to propranolol, a specific beta-AR antagonist, but abolished by prazosin, an alpha(1)-AR antagonist, suggesting that alpha(1)-AR but not beta-AR mediated the phosphorylation of AMPK. Moreover, the results from rat models of 7-day-infusion of AR agonists demonstrated that the activity of AMPK was significantly higher in NE (7.4-fold) and PE (6.0-fold) infusion groups than that in the vehicle group (P<0.05, n=6). On the other hand, no obvious cardiac hypertrophy and tissue fibrosis changes were observed in PE-infused rats. Taken together, our results demonstrate that alpha(1)-AR stimulation enhances the activity of AMPK, indicating an important role of alpha(1)-AR stimulation in the regulation of AMPK in the heart. Understanding the activation of AMPK mediated by alpha(1)-AR might have clinical implications in the therapy of heart failure.
