Design, Multigram Synthesis, and in Vitro and in Vivo Evaluation of Propylamycin: A Semisynthetic 4,5-Deoxystreptamine Class Aminoglycoside for the Treatment of Drug-Resistant Enterobacteriaceae and Other Gram-Negative Pathogens.

Infectious diseases due to multidrug-resistant pathogens, particularly carbapenem-resistant Enterobacteriaceae (CREs), present a major and growing threat to human health and society, providing an urgent need for the development of improved potent antibiotics for their treatment. We describe the design and development of a new class of aminoglycoside antibiotics culminating in the discovery of propylamycin. Propylamycin is a 4'-deoxy-4'-alkyl paromomycin whose alkyl substituent conveys excellent activity against a broad spectrum of ESKAPE pathogens and other Gram-negative infections, including CREs, in the presence of numerous common resistance determinants, be they aminoglycoside modifying enzymes or rRNA methyl transferases. Importantly, propylamycin is demonstrated not to be susceptible to the action of the ArmA resistance determinant whose presence severely compromises the action of plazomicin and all other 4,6-disubstituted 2-deoxystreptamine aminoglycosides. The lack of susceptibility to ArmA, which is frequently encoded on the same plasmid as carbapenemase genes, ensures that propylamycin will not suffer from problems of cross-resistance when used in combination with carbapenems. Cell-free translation assays, quantitative ribosome footprinting, and X-ray crystallography support a model in which propylamycin functions by interference with bacterial protein synthesis. Cell-free translation assays with humanized bacterial ribosomes were used to optimize the selectivity of propylamycin, resulting in reduced ototoxicity in guinea pigs. In mouse thigh and septicemia models of Escherichia coli, propylamycin shows excellent efficacy, which is better than paromomycin. Overall, a simple novel deoxy alkyl modification of a readily available aminoglycoside antibiotic increases the inherent antibacterial activity, effectively combats multiple mechanisms of aminoglycoside resistance, and minimizes one of the major side effects of aminoglycoside therapy.

Chemical probing for examining the structure of modified RNAs and ligand binding to RNA.

Among different RNA modifications, the helix 69 (H69) region of the bacterial ribosomal RNA (rRNA) contains three pseudouridines (Ψs). H69 is functionally important due to its location in the heart of the ribosome. Several structural and functional studies have shown the importance of Ψ modifications in influencing the H69 conformation as well as maintaining key interactions in the ribosome during protein synthesis. Therefore, a need exists to understand the influence of modified nucleosides on conformational dynamics of the ribosome under solution conditions that mimic the cellular environment. In this review on chemical probing, we provide detailed protocols for the use of dimethyl sulfate (DMS) to examine H69 conformational states and the influence of Ψ modifications under varying solution conditions in the context of both ribosomal subunits and full ribosomes. The use of DMS footprinting to study the binding of aminoglycosides to the H69 region of bacterial rRNA as a potential antibiotic target will also be discussed. As highlighted in this work, DMS probing and footprinting are versatile techniques that can be used to gain important insight into RNA local structure and RNA-ligand interactions, respectively.

FOXL2 is a sensitive and specific marker for sex cord-stromal tumors of the ovary.

Sex cord-stromal tumors (SCSTs) of the ovary are relatively uncommon tumors. Diagnosis of SCST rests primarily on the histomorphology of these tumors, and tumors with an atypical or unconventional appearance can pose diagnostic challenges. Previously, we had identified FOXL2 (402C→G) mutation as being characteristic of adult granulosa cell tumors (aGCTs). However, molecular screening for this mutation is not always possible and adds time and cost to the diagnostic process. In this study, we investigated the potential diagnostic use of immunostaining for FOXL2 on formalin-fixed paraffin-embedded tissue sections. Using a commercially available polyclonal antiserum against FOXL2 protein, immunoexpression of FOXL2 was tested in 501 ovarian tumor samples, including 119 SCSTs, using whole tissue sections and tissue microarrays. Staining was correlated with FOXL2 mutation status. In addition, we compared FOXL2 immunoexpression with that of α-inhibin and calretinin, the 2 traditional immunomarkers of SCST, in a subset of 89 SCSTs. FOXL2 immunostaining was present in 95 of 119 (80%) SCSTs, including >95% of aGCTs, juvenile granulosa cell tumors, fibromas, and sclerosing stromal tumors. Only 50% of Sertoli-Leydig cell tumors (N=40) expressed FOXL2. One of 11 steroid cell tumors and 3 of 3 female adnexal tumors of probable Wolffian origin showed FOXL2 immunoreactivity, whereas all other non-SCSTs tested (N=368) were negative for FOXL2 expression. Thus, the sensitivity and specificity of FOXL2 immunoreactivity for SCST are 80% and 99%, respectively. The FOXL2 (402C→G) mutation was confirmed to be both a sensitive and relatively specific indicator of aGCT. Forty-five of 119 SCSTs were mutation positive. These cases were 39 of 42 (93%) aGCTs, 3 of 40 Sertoli-Leydig cell tumors, 2 of 5 thecomas, and 1 of 4 (25%) SCSTs of unclassified type. SCSTs harboring a FOXL2 mutation consistently immunoexpressed FOXL2 (44 of 45, 98%), but FOXL2 immunostaining was also seen in many SCSTs that lacked a mutation (49 of 73, 67%). FOXL2 immunostaining showed higher sensitivity for the diagnosis of SCST, compared with α-inhibin and calretinin, and FOXL2 staining was typically more intense in positive cases compared with either α-inhibin or calretinin. In the SCSTs that were negative for FOXL2 expression, α-inhibin and/or calretinin immunostaining yielded positive results. In conclusion, FOXL2 is a relatively sensitive and highly specific marker for SCST. FOXL2 staining is present in almost all SCSTs with a FOXL2 mutation, and also in a majority of SCSTs without a mutation. FOXL2, together with α-inhibin and calretinin, forms an immunomarker panel that will result in positive staining with 1 or more markers in essentially all cases of SCST.