Translation inhibition by rocaglates activates a species-specific cell death program in the emerging fungal pathogen Candida auris

Fungal infections are a major contributor to infectious disease-related deaths worldwide. Recently, global emergence of the fungal pathogen Candida auris has caused considerable concern because most C. auris isolates are resistant to fluconazole, the most commonly administered antifungal, and some isolates are resistant to drugs from all three major antifungal classes. To identify novel agents with bioactivity against C. auris, we screened 2,454 compounds from a diversity-oriented synthesis collection. Of the five hits identified, most shared a common rocaglate core structure and displayed fungicidal activity against C. auris These rocaglate hits inhibited translation in C. auris but not in its pathogenic relative Candida albicans Species specificity was contingent on variation at a single amino acid residue in Tif1, a fungal member of the eukaryotic initiation factor 4A (eIF4A) family of translation initiation factors known to be targeted by rocaglates. Rocaglate-mediated inhibition of translation in C. auris activated a cell death program characterized by loss of mitochondrial membrane potential, increased caspase-like activity, and disrupted vacuolar homeostasis. In a rocaglate-sensitized C. albicans mutant engineered to express translation initiation factor 1 (Tif1) with the variant amino acid that we had identified in C. auris, translation was inhibited but no programmed cell death phenotypes were observed. This surprising finding suggests divergence between these related fungal pathogens in their pathways of cellular responses to translation inhibition. From a therapeutic perspective, the chemical biology that we have uncovered reveals species-specific vulnerability in C. auris and identifies a promising target for development of new, mechanistically distinct antifungals in the battle against this emerging pathogen. IMPORTANCE Emergence of the fungal pathogen Candida auris has ignited intrigue and alarm within the medical community and the public at large. This pathogen is unusually resistant to antifungals, threatening to overwhelm current management options. By screening a library of structurally diverse molecules, we found that C. auris is surprisingly sensitive to translation inhibition by a class of compounds known as rocaglates (also known as flavaglines). Despite the high level of conservation across fungi in their protein synthesis machinery, these compounds inhibited translation initiation and activated a cell death program in C. auris but not in its relative Candida albicans Our findings highlight a surprising divergence across the cell death programs operating in Candida species and underscore the need to understand the specific biology of a pathogen in attempting to develop more-effective treatments against it.Published versio

Surface Grafting of Poly(L-glutamates). 3. Block Copolymerization

This paper describes for the first time the synthesis of surface-grafted AB-block copolypeptides, consisting of poly(γ-benzyl L-glutamate) (PBLG) as the A-block and poly(γ-methyl L-glutamate) (PMLG) as the B-block. Immobilized primary amine groups of (γ-aminopropyl)triethoxysilane (APS) on silicon wafers initiated the ring-opening polymerization of N-carboxyanhydrides of glutamic acid esters (NCAs). After removal of the BLG-NCA monomer solution after a certain reaction time, the amine end groups of the formed PBLG blocks acted as initiators for the second monomers. This method provides the possibility of making layered structures of surface-grafted block copolymers with tuned properties. Ellipsometry and small-angle X-ray reflection (SAXR) measurements revealed the thickness of the polypeptide layers ranging from 45-100 Å of the first block to 140-270 Å for the total block copolypeptides. The chemical composition of the blocks was determined by X-ray photoelectron spectroscopy (XPS). In addition, Fourier transform infrared transmission spectroscopy (FT-IR) revealed that the polypeptide main chains of both blocks consisted of pure R-helices. The average orientation of the helices ranging from 22-42° with respect to the substrate within the first block to 31-35° in the second block could be derived with FT-IR as well.

Surface Grafting of Poly(L-glutamates). 2. Helix Orientation

In this paper the average helix orientation of surface-grafted poly(γ-benzyl L-glutamate) (PBLG), poly(γ-methyl L-glutamate) (PMLG), and poly(γ-methyl L-glutamate)-co-(γ-n-stearyl L-glutamate) (PMLGSLG 70/30) was investigated by means of FT-IR transmission spectroscopy. The theoretical relation between the average tilt angle (θ) and the absorption peak areas of three different backbone amide bands could be calculated because their transition dipole moment directions with respect to the helix axis were known. From the normalized absorptions, the average tilt angles of grafted helices of PBLG, PMLG, and PMLGSLG 70/30 were determined. The somewhat larger average angle of PMLG helices of 35 ± 5° with respect to the substrate compared to the value of 32 ± 5° of PBLG was due to the higher grafting density of PMLG. Because of the smaller helix diameter as a result of the smaller size of the methyl side group, more PMLG helices grew on the same surface area. Sterical hindrance and unfavorable polar interactions between unidirectional aligned helices forced the PMLG helices in a more upright arrangement. The even more perpendicular orientation of PMLGSLG 70/30 (48 ± 6°) could be the result of incorporation of mainly γ-methyl L-glutamate N-carboxyanhydride (MLG-NCA) monomers during the initiation step. Incorporation of the much larger γ-n-stearyl L-glutamate N-carboxyanhydride (SLG-NCA) monomers afterward lead to enlarged angles with respect to the substrate. Due to swelling, a pronounced change in helix orientation of grafted PMLGSLG 70/30 in n-hexadecane was observed, resulting in an almost perpendicular helix orientation.

Malaria surveillance in the Democratic Republic of the Congo: comparison of microscopy, PCR, and rapid diagnostic test

Malaria surveillance is critical for control efforts, but diagnostic methods frequently disagree. Here we compare microscopy, PCR, and a Rapid Diagnostic Test in 7,137 samples from children in the Democratic Republic of the Congo using Latent Class Analysis. PCR had the highest sensitivity (94.6%) and microscopy had the lowest (76.7%)

Low prevalence of Plasmodium malariae and Plasmodium ovale mono-infections among children in the Democratic Republic of the Congo: a population-based, cross-sectional study

Abstract Background In an effort to improve surveillance for epidemiological and clinical outcomes, rapid diagnostic tests (RDTs) have become increasingly widespread as cost-effective and field-ready methods of malaria diagnosis. However, there are concerns that using RDTs specific to Plasmodium falciparum may lead to missed detection of other malaria species such as Plasmodium malariae and Plasmodium ovale. Methods Four hundred and sixty six samples were selected from children under 5 years old in the Democratic Republic of the Congo (DRC) who took part in a Demographic and Health Survey (DHS) in 2013–14. These samples were first tested for all Plasmodium species using an 18S ribosomal RNA-targeted real-time PCR; malaria-positive samples were then tested for P. falciparum, P. malariae and P. ovale using a highly sensitive nested PCR. Results The prevalence of P. falciparum, P. malariae and P. ovale were 46.6, 12.9 and 8.3 %, respectively. Most P. malariae and P. ovale infections were co-infected with P. falciparum—the prevalence of mono-infections of these species were only 1.0 and 0.6 %, respectively. Six out of these eight mono-infections were negative by RDT. The prevalence of P. falciparum by the more sensitive nested PCR was higher than that found previously by real-time PCR. Conclusions Plasmodium malariae and P. ovale remain endemic at a low rate in the DRC, but the risk of missing malarial infections of these species due to falciparum-specific RDT use is low. The observed prevalence of P. falciparum is higher with a more sensitive PCR method

Synthesis and structural characterization of a mimetic membrane-anchored prion protein

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrPSc) of the host encoded prion protein (PrPC) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrPC and PrPSc have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrPC to PrPSc, but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP

Safety of ECT in patients receiving an oral anticoagulant

Introduction: This study assessed the use, tolerability, and safety of anticoagulation via direct oral anticoagulants or warfarin in medical and psychiatric inpatients receiving ECT. Methods: This retrospective cohort study included 32 patients who received ECT while on either a direct oral anticoagulant (9) or warfarin (23) and spanned 247 encounters at Maine Medical Center between December 2012 and December 2018. Data are presented descriptively and analyzed using SPSS version 25 and Microsoft Excel version 2016. Results: Among the 247 ECT patient encounters, there were few major adverse effects of ECT in this medically complex population. These adverse effects included headache during 4 encounters (1.6%), respiratory distress during 2 encounters (0.8%) and a cardiovascular event during 1 encounter (0.4%). One patient (3.1%) who was receiving concurrent rivaroxaban and venlafaxine experienced gastrointestinal bleeding that was determined to be unrelated to ECT. One patient on fluoxetine and warfarin experienced hemoptysis thought to be secondary to epistaxis. No other major bleeding or clotting event occurred during an ECT session nor for the duration of the hospitalization. Discussion: Direct oral anticoagulants and warfarin appear safe in the treatment of patients with atrial fibrillation or acute venous thromboembolism who are receiving concomitant ECT. Prospective studies are needed to confirm these findings

Latent class analysis of the Child Behavior Checklist Obsessive-Compulsive Scale

The Obsessive-Compulsive Scale (OCS) of the Child Behavior Checklist (CBCL) predicts obsessive-compulsive disorder and is highly heritable. Latent class analysis (LCA) of the OCS was used to identify profiles within this 8-item scale and to examine heritability of those profiles. The LCA was performed on maternal CBCL reports of their 6- to 18-year-old children from 2 US nationally representative samples from 1989 (n = 2475, 50% male) and 1999 (n = 2029, 53% male) and from Dutch twins in the Netherlands Twin Registry at ages 7 (n = 10 194, 49.3% male), 10 (n = 6448, 48.1% male), and 12 (n = 3674, 48.6% male) years. The heritability of the resultant classes was estimated using odds ratios of twin membership across classes. A 4-class solution fitted all samples best. The resulting classes were a "No or Few Symptoms" class, a "Worries and Has to Be Perfect" class, a "Thought Problems" class, and an "OCS" class. Within-class odds ratios were higher than across-class odds ratios and were higher for monozygotic than dizygotic twins. We conclude that LCA identifies an OCS class and that class is highly heritable using across-twin comparisons. © 2009 Elsevier Inc. All rights reserved