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Moringin (rhamnobenzyl isothiocyanate) is a major bioactive compound in moringa seeds, which have been used as a healthy food. [https://www.selleckchem.com/products/shin1-rz-2994.html https://www.selleckchem.com/products/shin1-rz-2994.html] However, its bioactivity mechanisms are not well understood. We investigated moringin and its structurally similar analogues, including benzyl isothiocyanate and 4-hydroxylbenzyl isothiocyanate, for their hydrogen sulfide (H2S)-releasing activity triggered by cysteine. These isothiocyanates rapidly formed cysteine adducts, which underwent intramolecular cyclization followed by slowly releasing (a) organic amine and raphanusamic acid and (b) H2S and 2-carbylamino-4,5-dihydrothiazole-4-carboxylic acids. The product distributions are highly dependent on para-substituents on the phenyl group. Moringin has higher cytotoxicity to cancer cells and is a more potent anti-inflammatory agent than benzyl and hydroxybenzyl analogues, while benzyl isothiocyanate is a better antibacterial agent. Taken together, their bioactivity may not be directly related to their H2S donation activity. However, other metabolites alone do not have cytotoxicity and anti-inflammatory activity. These findings indicated that their activity may be the combination effects of different metabolites via competitive pathways as well the para-substituent groups of benzyl ITCs.Hydrogels of biopolymers such as agar and gelatin are widely used in many applications, and in many cases, the gels are loaded with nanoparticles. The polymer chains in these gels are cross-linked by physical bonds into three-dimensional networks, with the mesh size of these networks typically being 10-100 nm. One class of "soft" nanoparticles are liposomes, which have an aqueous core surrounded by a lipid bilayer. Solutes encapsulated in the liposomal core can be delivered externally over time. In this paper, we create liposomes with diameters ∼150 nm from an unsaturated phospholipid (lecithin) and embed them in agar gels (the aqueous phase also contains 0-50% of glycerol, which is an active ingredient in cosmetic products). Upon placing this gel in quiescent water, we find that the liposomes release out of the gel into the water over a period of 1-3 days, even though the gel remains intact. This is a surprising result that runs contrary to our expectation that the liposomes would simply remain immobilized in the gel. We show that the release rate of liposomes can be tuned by several variables for example, the release rate increases as the agar concentration is lowered and the rate increases steadily with temperature. In addition to agar, release of liposomes also occurs out of other physical gels including those of agarose and gelatin. However, liposomes made from a saturated phospholipid do not release out of any gels. We discuss a possible mechanism for liposomal release, which involves intact liposomes deforming and squeezing through transient large pores that arise in physical networks such as agar. Our findings have relevance to transdermal delivery they suggest the possibility of systematically delivering liposomes loaded with actives out of an intact matrix.Cytosine methylation is an important epigenetic mark, but how the distinctive patterns of DNA methylation arise remains elusive. For the first time, we systematically investigated how these patterns can be imparted by the inherent enzymatic preferences of mammalian de novo DNA methyltransferases in vitro and the extent to which this applies in cells. In a biochemical experiment, we subjected a wide variety of DNA sequences to methylation by DNMT3A or DNMT3B and then applied deep bisulfite sequencing to quantitatively determine the sequence preferences for methylation. The data show that DNMT3A prefers CpG and non-CpG sites followed by a 3'-pyrimidine, whereas DNMT3B favors a 3'-purine. Overall, we show that DNMT3A has a sequence preference for a TNC[G/A]CC context, while DNMT3B prefers TAC[G/A]GC. We extended our finding using publicly available data from mouse Dnmt1/3a/3b triple-knockout cells in which reintroduction of either DNMT3A or DNMT3B expression results in the acquisition of the same enzyme specific signature sequences observed in vitro. Furthermore, loss of DNMT3A or DNMT3B in human embryonic stem cells leads to a loss of methylation at the corresponding enzyme specific signatures. Therefore, the global DNA methylation landscape of the mammalian genome can be fundamentally determined by the inherent sequence preference of de novo methyltransferases.Recently, we heterologously expressed, purified, and analyzed the function of the sole Plasmodium falciparum phosphatidylinositol 3-kinase (PI3K), found that the enzyme is a "class III" or "Vps34" PI3K, and found that it is irreversibly inhibited by Fe2+-mediated covalent, nonspecific interactions with the leading antimalarial drug, dihydroartemisinin [Hassett, M. R., et al. (2017) Biochemistry 56, 4335-4345]. One of several P. falciparum phosphatidylinositol 4-kinases [putative IIIβ isoform (PfPI4KIIIβ)] has generated similar interest as a druggable target; however, no validation of the mechanism of action for putative PfPI4K inhibitors has yet been possible due to the lack of purified PfPI4KIIIβ. We therefore codon optimized the pfpi4kIIIβ gene, successfully expressed the protein in yeast, and purified an N-lobe catalytic domain PfPI4KIIIβ protein. Using an enzyme-linked immunosorbent assay strategy previously perfected for analysis of PfPI3K (PfVps34), we measured the apparent initial rate, Km,app(ATP), and other enzyme characteristics and found full activity for the construct and that PfPI4KIIIβ activity is most consistent with the class IIIβ designation. Because several novel antimalarial drug candidates with different chemical scaffolds have been proposed to target PfPI4KIIIβ, we titrated enzyme inhibition for these candidates versus purified PfPI4KIIIβ and PfVps34. We also analyzed the activity versus purified PfPI4KIIIβ mutants previously expressed in P. falciparum selected for resistance to these drugs. Interestingly, we found that a putative PfPI4KIIIβ inhibitor currently in advanced trials (MMV390048; MMV '0048) is a potent inhibitor of both PfVps34 and PfPI4KIIIβ. These data are helpful for further preclinical optimization of an exciting new class of P. falciparum PI kinase inhibitor ("PfPIKi") antimalarial drugs.