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.

Neuronal glutamate transporters control reciprocal inhibition and gain modulation in D1 medium spiny neurons.

Understanding the function of glutamate transporters has broad implications for explaining how neurons integrate information and relay it through complex neuronal circuits. Most of what is currently known about glutamate transporters, specifically their ability to maintain glutamate homeostasis and limit glutamate diffusion away from the synaptic cleft, is based on studies of glial glutamate transporters. By contrast, little is known about the functional implications of neuronal glutamate transporters. The neuronal glutamate transporter EAAC1 is widely expressed throughout the brain, particularly in the striatum, the primary input nucleus of the basal ganglia, a region implicated with movement execution and reward. Here, we show that EAAC1 limits synaptic excitation onto a population of striatal medium spiny neurons identified for their expression of D1 dopamine receptors (D1-MSNs). In these cells, EAAC1 also contributes to strengthen lateral inhibition from other D1-MSNs. Together, these effects contribute to reduce the gain of the input-output relationship and increase the offset at increasing levels of synaptic inhibition in D1-MSNs. By reducing the sensitivity and dynamic range of action potential firing in D1-MSNs, EAAC1 limits the propensity of mice to rapidly switch between behaviors associated with different reward probabilities. Together, these findings shed light on some important molecular and cellular mechanisms implicated with behavior flexibility in mice.

Estimating the glutamate transporter surface density in distinct sub-cellular compartments of mouse hippocampal astrocytes.

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes, and previous estimates have been obtained about their surface expression in astrocytes of the rat hippocampus and cerebellum. Analogous estimates for the mouse hippocampus are currently not available. In this work, we derive the surface density of astrocytic glutamate transporters in mice of different ages via quantitative dot blot. We find that the surface density of glial glutamate transporters is similar in 7-8 week old mice and rats. In mice, the levels of glutamate transporters increase until about 6 months of age and then begin to decline slowly. Our data, obtained from a combination of experimental and modeling approaches, point to the existence of stark differences in the density of expression of glutamate transporters across different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on their level of synaptic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on how heterogeneity in the spatial distribution of glutamate transporters in the plasma membrane of hippocampal astrocytes my alter glutamate receptor activation out of the synaptic cleft.

Circadian Modulation of Neurons and Astrocytes Controls Synaptic Plasticity in Hippocampal Area CA1.

Most animal species operate according to a 24-h period set by the suprachiasmatic nucleus (SCN) of the hypothalamus. The rhythmic activity of the SCN modulates hippocampal-dependent memory, but the molecular and cellular mechanisms that account for this effect remain largely unknown. Here, we identify cell-type-specific structural and functional changes that occur with circadian rhythmicity in neurons and astrocytes in hippocampal area CA1. Pyramidal neurons change the surface expression of NMDA receptors. Astrocytes change their proximity to synapses. Together, these phenomena alter glutamate clearance, receptor activation, and integration of temporally clustered excitatory synaptic inputs, ultimately shaping hippocampal-dependent learning in vivo. We identify corticosterone as a key contributor to changes in synaptic strength. These findings highlight important mechanisms through which neurons and astrocytes modify the molecular composition and structure of the synaptic environment, contribute to the local storage of information in the hippocampus, and alter the temporal dynamics of cognitive processing.

Discovery of Small-Molecule Antibiotics against a Unique tRNA-Mediated Regulation of Transcription in Gram-Positive Bacteria.

The emergence of multidrug-resistant bacteria necessitates the identification of unique targets of intervention and compounds that inhibit their function. Gram-positive bacteria use a well-conserved tRNA-responsive transcriptional regulatory element in mRNAs, known as the T-box, to regulate the transcription of multiple operons that control amino acid metabolism. T-box regulatory elements are found only in the 5'-untranslated region (UTR) of mRNAs of Gram-positive bacteria, not Gram-negative bacteria or the human host. Using the structure of the 5'UTR sequence of the Bacillus subtilis tyrosyl-tRNA synthetase mRNA T-box as a model, in silico docking of 305 000 small compounds initially yielded 700 as potential binders that could inhibit the binding of the tRNA ligand. A single family of compounds inhibited the growth of Gram-positive bacteria, but not Gram-negative bacteria, including drug-resistant clinical isolates at minimum inhibitory concentrations (MIC 16-64 µg mL-1 ). Resistance developed at an extremely low mutational frequency (1.21×10-10 ). At 4 µg mL-1 , the parent compound PKZ18 significantly inhibited in vivo transcription of glycyl-tRNA synthetase mRNA. PKZ18 also inhibited in vivo translation of the S. aureus threonyl-tRNA synthetase protein. PKZ18 bound to the Specifier Loop in vitro (Kd ≈24 µm). Its core chemistry necessary for antibacterial activity has been identified. These findings support the T-box regulatory mechanism as a new target for antibiotic discovery that may impede the emergence of resistance.

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.

Evidence in support of RNA-mediated inhibition of phosphatidylserine-dependent HIV-1 Gag membrane binding in cells.

The matrix domain promotes plasma-membrane-specific binding of HIV-1 Gag through interaction with an acidic lipid phosphatidylinositol-(4,5)-bisphosphate. In in vitro systems, matrix-bound RNA suppresses Gag interactions with phosphatidylserine, an acidic lipid prevalent in various cytoplasmic membranes, thereby enhancing the lipid specificity of the matrix domain. Here we provide in vitro and cell-based evidence supporting the idea that this RNA-mediated suppression occurs in cells and hence is a physiologically relevant mechanism that prevents Gag from binding promiscuously to phosphatidylserine-containing membranes.

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.