In Vitro Activities of the Novel Investigational Tetrazoles VT-1161 and VT-1598 Compared to the Triazole Antifungals against Azole-Resistant Strains and Clinical Isolates of Candida albicans

The fungal Cyp51-specific inhibitors VT-1161 and VT-1598 have emerged as promising new 24 therapies to combat fungal infections, including Candida spp. To evaluate the in vitro activity of 25 these compounds in comparison to other available azoles, minimum inhibitory concentrations 26 (MICs) were determined for VT-1161, VT-1598, fluconazole, voriconazole, itraconazole, and 27 posaconazole against 68 C. albicans clinical isolates well-characterized for azole resistance 28 mechanisms and mutant strains representing individual azole resistance mechanisms. VT-1161 29 and VT-1598 demonstrated potent activity (geometric mean MICs ≤0.15 μg/mL) against 30 predominantly fluconazole-resistant isolates. However, five of 68 isolates exhibited MICs 31 greater than six dilutions (>2 μg/mL) to both tetrazoles compared to fluconazole-susceptible 32 isolates. Four of these isolates likewise exhibited high MICs beyond the upper limit for all 33 triazoles tested. A premature stop codon in ERG3 likely explained the high-level resistance in 34 one isolate. VT-1598 was effective against strains with hyperactive Tac1, Mrr1, and Upc2 35 transcription factors and against most ERG11 mutant strains. VT-1161 MICs were elevated for 36 hyperactive Tac1 strains and two strains with Erg11 substitutions (Y132F and Y132F&K143R), 37 but showed activity against strains with hyperactive forms of Mrr1 and Upc2. VT-1161 had 38 elevated MICs against a minority of clinical isolates that were more susceptible to itraconazole 39 (3), voriconazole (1), or posaconazole (5). While mutations affecting Erg3 activity appear to 40 greatly reduce susceptibility to VT-1161 and VT-1598, the elevated MICs of both tetrazoles for 41 four isolates could not be explained by known azole resistance mechanisms, suggesting the 42 presence of undescribed resistance mechanisms to triazole- and tetrazole-based sterol 43 demethylase inhibitors

Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana

Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites’ genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51). The mutation, N176I, is found outside of the enzyme’s active site, consistent with the fact that the resistant line continues to produce the enzyme’s product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself

Improved functionalization of oleic acid-coated iron oxide nanoparticles for biomedical applications

Superparamagnetic iron oxide nanoparticles can providemultiple benefits for biomedical applications in aqueous environments such asmagnetic separation or magnetic resonance imaging. To increase the colloidal stability and allow subsequent reactions, the introduction of hydrophilic functional groups onto the particles’ surface is essential. During this process, the original coating is exchanged by preferably covalently bonded ligands such as trialkoxysilanes. The duration of the silane exchange reaction, which commonly takes more than 24 h, is an important drawback for this approach. In this paper, we present a novel method, which introduces ultrasonication as an energy source to dramatically accelerate this process, resulting in high-quality waterdispersible nanoparticles around 10 nmin size. To prove the generic character, different functional groups were introduced on the surface including polyethylene glycol chains, carboxylic acid, amine, and thiol groups. Their colloidal stability in various aqueous buffer solutions as well as human plasma and serum was investigated to allow implementation in biomedical and sensing applications.status: publishe

5-Formylcytosine to Cytosine Conversion by C-C Bond Cleavage in vivo

Tet enzymes oxidise 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA. It was proposed that fdC and cadC deformylate and decarboxylate to dC in the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites. The question whether such direct C-C bond cleavage reactions at fdC and cadC occur in vivo remains an unsolved problem. Here we report the incorporation of synthetic isotope- and (R)-2’-fluorine-labelled dC and fdC-derivatives into the genome of cultured mammalian cells. Following the fate of these probe molecules using UHPLC-MS/MS provided quantitative data about the formed reaction products. The data show that the labelled fdC probe is efficiently converted into the corresponding labelled dC, most likely after its incorporation into the genome. This allows concluding that fdC is undergoing C-C bond cleavage in stem cells that leads to the direct re-installation of unmodified dC