Clinical Course, Antifungal Susceptibility, and Genomic Sequencing of Trichophyton indotineae.

Importance: Trichophyton indotineae is an emerging dermatophyte causing outbreaks of extensive tinea infections often unresponsive to terbinafine. This species has been detected worldwide and in multiple US states, yet detailed US data on infections with T indotineae are sparse and could improve treatment practices and medical understanding of transmission. Objective: To correlate clinical features of T indotineae infections with in vitro antifungal susceptibility testing results, squalene epoxidase gene sequence variations, and isolate relatedness using whole-genome sequencing. Design, Setting, and Participants: This retrospective cohort study of patients with T indotineae infections in New York City spanned May 2022 to May 2023. Patients with confirmed T indotineae infections were recruited from 6 New York City medical centers. Main Outcome and Measure: Improvement or resolution at the last follow-up assessment. Results: Among 11 patients with T indotineae (6 male and 5 female patients; median [range] age, 39 [10-65] years), 2 were pregnant; 1 had lymphoma; and the remainder were immunocompetent. Nine patients reported previous travel to Bangladesh. All had widespread lesions with variable scale and inflammation, topical antifungal monotherapy failure, and diagnostic delays (range, 3-42 months). Terbinafine treatment failed in 7 patients at standard doses (250 mg daily) for prolonged duration; these patients also had isolates with amino acid substitutions at positions 393 (L393S) or 397 (F397L) in squalene epoxidase that correlated with elevated terbinafine minimum inhibitory concentrations of 0.5 µg/mL or higher. Patients who were treated with fluconazole and griseofulvin improved in 2 of 4 and 2 of 5 instances, respectively, without correlation between outcomes and antifungal minimum inhibitory concentrations. Furthermore, 5 of 7 patients treated with itraconazole cleared or had improvement at the last follow-up, and 2 of 7 were lost to follow-up or stopped treatment. Based on whole-genome sequencing analysis, US isolates formed a cluster distinct from Indian isolates. Conclusion and Relevance: The results of this case series suggest that disease severity, diagnostic delays, and lack of response to typically used doses and durations of antifungals for tinea were common in this primarily immunocompetent patient cohort with T indotineae, consistent with published data. Itraconazole was generally effective, and the acquisition of infection was likely in Bangladesh.

Inhibition of HIV-1 Gag-membrane interactions by specific RNAs.

HIV-1 particle assembly, which occurs at the plasma membrane (PM) of cells, is driven by the viral polyprotein Gag. Gag recognizes phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], a PM-specific phospholipid, via the highly basic region (HBR) in its N-terminal matrix (MA) domain. The HBR is also known to bind to RNA. We have previously shown, using an in vitro liposome binding assay, that RNA inhibits Gag binding to membranes that lack PI(4,5)P2 If this RNA block is removed by RNase treatment, Gag can bind nonspecifically to other negatively charged membranes. In an effort to identify the RNA species that confer this inhibition of Gag membrane binding, we have tested the impact of purified RNAs on Gag interactions with negatively charged liposomes lacking PI(4,5)P2 We found that some tRNA species and RNAs containing stem-loop 1 of the psi region in the 5' untranslated region of the HIV-1 genome impose inhibition of Gag binding to membranes lacking PI(4,5)P2 In contrast, a specific subset of tRNAs, as well as an RNA sequence previously selected in vitro for MA binding, failed to suppress Gag-membrane interactions. Furthermore, switching the identity of charged residues in the HBR did not diminish the susceptibility of Gag-liposome binding for each of the RNAs tested, while deletion of most of the NC domain abrogates the inhibition of membrane binding mediated by the RNAs that are inhibitory to WT Gag-liposome binding. These results support a model in which NC facilitates binding of RNA to MA and thereby promotes RNA-based inhibition of Gag-membrane binding.

Methods to Study Determinants for Membrane Targeting of HIV-1 Gag In Vitro.

Assembly of HIV-1 viral particles is a critical step of the HIV-1 life cycle; yet many details of this complex process are unknown. The Gag polyprotein drives viral particle assembly at the plasma membrane via three different types of interactions: protein-protein, protein-RNA, and protein-membrane interactions. As an approach to tease apart the importance of these interactions during viral particle assembly, in particular at the step of Gag membrane binding, we have developed an in vitro liposome-binding assay. Below we describe how to prepare liposomes, which serve as model membranes, and how to assess their interaction with Gag by liposome flotation centrifugation. Additionally, we outline extensions of this basic assay that can be used to address the role of RNA in regulating Gag-membrane interactions.

Secondary structure of bacteriophage T4 gene 60 mRNA: implications for translational bypassing.

Translational bypassing is a unique phenomenon of bacteriophage T4 gene 60 mRNA wherein the bacterial ribosome produces a single polypeptide chain from a discontinuous open reading frame (ORF). Upon reaching the 50-nucleotide untranslated region, or coding gap, the ribosome either dissociates or bypasses the interruption to continue translating the remainder of the ORF, generating a subunit of a type II DNA topoisomerase. Mutational and computational analyses have suggested that a compact structure, including a stable hairpin, forms in the coding gap to induce bypassing, yet direct evidence is lacking. Here we have probed the secondary structure of gene 60 mRNA with both Tb³âº ions and the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) reagent 1M7 under conditions where bypassing is observed. The resulting experimentally informed secondary structure models strongly support the presence of the predicted coding gap hairpin and highlight the benefits of using Tb³âº as a second, complementary probing reagent. Contrary to several previously proposed models, however, the rest of the coding gap is highly reactive with both probing reagents, suggesting that it forms only a short stem-loop. Mutational analyses coupled with functional assays reveal that two possible base-pairings of the coding gap with other regions of the mRNA are not required for bypassing. Such structural autonomy of the coding gap is consistent with its recently discovered role as a mobile genetic element inserted into gene 60 mRNA to inhibit cleavage by homing endonuclease MobA.

Characterization of nucleic acid higher order structure by gas-phase H/D exchange in a quadrupole-FT-ICR mass spectrometer.

Nucleic acid higher order structure is of intense interest in antisense and antigene strategies toward novel chemotherapeutic agents. Understanding how structural characteristics affect solution-phase properties is essential for a rational approach to nucleic acid-targeted drug design. The most dominant nucleic acid secondary structure is the hairpin, formed by intrastrand hydrogen bonding between complementary nucleobases. We have previously applied gas-phase hydrogen/deuterium exchange (HDX) with mass spectrometry detection to show that anionic DNA duplexes have lower HDX rates than their constituent monomers, indicating that hydrogen bonding can shield hydrogens from exchanging with the bath gas D(2)S. The same HDX assay is applied here to investigate nucleic acid hairpin structure. Variations in hairpin solution-phase stabilities are achieved by changing their loop size, stem length, and stem composition (ratio of G/C and A/T(U) base pairs in the stem). These differences can be carried into the gas phase because electrospray ionization is a gentle ionization method that is able to preserve noncovalent interactions. Observed gas-phase HDX rates of these hairpins are consistent with their relative solution-phase stabilities as predicted by MFold, i.e., less stable nucleic acid hairpins exchange faster than more stable hairpins. To our knowledge, the presented experiments demonstrate for the first time that gas-phase HDX may be used to characterize nucleic acid higher order structure and the results suggest that the relative stabilities of nucleic acid hairpins in the gaseous phase are correlated with those in solution.