Jerniganroy1399

Quantum dots (QDs) are nanocrystals with bright fluorescence and long-term photostability, attributes particularly beneficial for single-molecule imaging and molecular counting in the life sciences. The size of a QD nanocrystal determines its physicochemical and photophysical properties, which dictate the success of imaging applications. Larger nanocrystals typically have better optical properties, with higher brightness, red-shifted emission, reduced blinking, and greater stability. However, larger nanocrystals introduce molecular labeling biases due to steric hindrance and nonspecific binding. Here we systematically analyze the impact of nanocrystal size on receptor labeling in live and fixed cells. We designed three (core)shell QDs with red emission (600-700 nm) and crystalline sizes of 3.2 nm, 5.5 nm, and 8.3 nm. After coating with the same multidentate polymer, hydrodynamic sizes were 9.2 nm (QD9.2), 13.3 nm (QD13.3), and 17.4 nm (QD17.4), respectively. The QDs were conjugated to streptavidin and applied as probes for biotinylated neurotransmitter receptors. QD9.2 exhibited the highest labeling specificity for receptors in the narrow synaptic cleft (~20-30 nm) in living neurons. However, for dense receptor labeling for molecular counting in live and fixed HeLa cells, QD13.3 yielded the highest counts. Nonspecific binding rose sharply for hydrodynamic sizes larger than 13.3 nm, with QD17.4 exhibiting particularly diminished specificity. Our comparisons further highlight needs to continue engineering the smallest QDs to increase single-molecule intensity, suppress blinking frequency, and inhibit nonspecific labeling in fixed and permeabilized cells. These results lay a foundation for designing QD probes with further reduced sizes to achieve unbiased labeling for quantitative and single-molecule imaging.Cytochrome (cyt) P460 is a c-type monoheme enzyme found in ammonia-oxidizing bacteria (AOB) and methanotrophs; additionally, genes encoding it have been found in some pathogenic bacteria. Cyt P460 is defined by a unique post-translational modification to the heme macrocycle, where a lysine (Lys) residue covalently attaches to the 13' meso carbon of the porphyrin, modifying this heme macrocycle into the enzyme's eponymous P460 cofactor, similar to the cofactor found in the enzyme hydroxylamine oxidoreductase. This cross-link imbues the protein with unique spectroscopic properties, the most obvious of which is the enzyme's green color in solution. Cyt P460 from the AOB Nitrosomonas europaea is a homodimeric redox enzyme that produces nitrous oxide (N2O) from 2 equiv of hydroxylamine. Mutation of the Lys cross-link results in spectroscopic features that are more similar to those of standard cyt c' proteins and renders the enzyme catalytically incompetent for NH2OH oxidation. Recently, the necessity of a second-sphere glutamate (Glu) residue for redox catalysis was established; it plausibly serves as proton relay during the first oxidative half of the catalytic cycle. Herein, we report the first crystal structure of a cross-link deficient cyt P460. This structure shows that the positioning of the catalytically essential Glu changes by approximately 0.8 Å when compared to a cross-linked, catalytically competent cyt P460. It appears that the heme-Lys cross-link affects the relative position of the P460 cofactor with respect to the second-sphere Glu residue, therefore dictating the catalytic competency of the enzyme.Combinatorial biosynthesis has great potential for designing synthetic circuits and amplifying the production of new active compounds. Studies on multienzyme cascades are extremely useful for improving our knowledge on enzymatic catalysis. In particular, the elucidation of enzyme substrate promiscuity can be potentially used for bioretrosynthetic approaches, leading to the design of alternative and more convenient routes to produce relevant molecules. In this perspective, plant-derived polyketides are extremely adaptable to those synthetic biological applications. Here, we present a combination of an in vitro CoA ligase activity assay coupled with a bacterial multigene expression system that leads to precursor-directed biosynthesis of 21 flavonoid derivatives. When the vast knowledge from protein databases is exploited, the herein presented procedure can be easily repeated with additional plant-derived polyketides. Lastly, we report an efficient in vivo expression system that can be further exploited to heterologously express pathways not necessarily related to plant polyketide synthases.The ProTide approach has emerged as a powerful tool to improve the intracellular delivery of nucleotide analogs with antiviral and anticancer activity. Here, we characterized the anti-ZIKV (ZIKV, Zika virus) activity of two ProTides of 2'-C-β-methylguanosine. ProTide UMN-1001 is a 2'-C-β-methylguanosine tryptamine phosphoramidate monoester, and ProTide UMN-1002 is a 2-(methylthio)-ethyl-2'-C-β-methylguanosine tryptamine phosphoramidate diester. UMN-1002 undergoes stepwise intracellular activation to the corresponding nucleotide monophosphate followed by P-N bond cleavage by intracellular histidine triad nucleotide binding protein 1 (Hint1). UMN-1001 is activated by Hint1 but is less cell-permeable than UMN-1002. UMN-1001 and UMN-1002 were found to be more potent than 2'-C-β-methylguanosine against ZIKV in human-derived microvascular endothelial and neuroblastoma cells and in reducing ZIKV RNA replication. Studies with a newborn mouse model of ZIKV infection demonstrated that, while treatment with 2'-C-β-methylguanosine and UMN-1001 was lethal, treatment with UMN-1002 was nontoxic and significantly reduced ZIKV infection. Our data suggests that anchimeric activated ProTides of 2'-C-β-methyl nucleosides should be further investigated for their potential as anti-ZIKV therapeutics.The hazard of hexavalent chromium (Cr(VI)) from environmental pollution and medical implanted metal has been recognized widely. However, removal of trace amount of Cr(VI) in the blood circumstance faces tremendous difficulties for that most of Cr(VI) located in erythrocytes, thus there is almost no literature to report the removal of Cr(VI) in blood. Herein, a removal strategy, named as reduction-adsorption-separation, is proposed to realize the removal of Cr(VI) in blood. First, magnetic core-shell mesoporous nanocomposite is fabricated by using Fe3O4 nanoparticles as magnetic core and mesoporous silica (MS) as shell, hyperbranched polyamide (HPA) as mesoporous channel modifier and ascorbic acid (ASC) as the reductant drug loaded in the mesoporous channels, which is also denoted as Fe/MS/HPA/ASC. RBPJ Inhibitor-1 clinical trial Then, on the basis of the bionic idea, the erythrocyte membrane (EM)-wrapped Fe/MS/HPA/ASC to protect ASC from deactivation is obtained and named as the therapeutic agent (Fe/MS/HPA/ASC@EM). During removal process, the therapeutic agent can enter in erythrocytes to use ASC to reduce Cr(VI) to Cr(III) and HPA in mesoporous channels to adsorb Cr(III) and can then be recollected from blood by magnetic separation.