Disparities in auditory physiology and pathology between C57BL/6J and C57BL/6N substrains.

C57BL/6 inbred mice are frequently used as models in auditory research, mostly the C57BL/6J and C57BL/6N substrains. Genetic variation and phenotypic disparities between these two substrains have been extensively investigated, but conflicting information exists about differences in their auditory and vestibular phenotypes. Literature-based comparisons are rendered difficult or impossible because most auditory publications do not designate the substrain used. We therefore evaluated commercial C57BL/6N and C57BL/6J mice for their baseline auditory brainstem response (ABR) thresholds at 3 months of age as well as their susceptibility to noise exposure and aminoglycoside antibiotics. Both substrains have similar thresholds at 4 and 12 kHz, but C57BL/6N show significantly higher baseline thresholds at 24 and 32 kHz. Because of these elevated thresholds, the N substrain is unsuitable as a model for drug ototoxicity, which primarily affects high frequencies. Exposure to 2-20 kHz broadband noise for 2 h at 110 dB produced significantly higher threshold shifts in the J substrain. These results suggest caution in the selection of C57BL/6 substrains for auditory research and indicate the need to specify substrains, age and the breeding source in all publications.

Identification and evaluation of improved 4'-O-(alkyl) 4,5-disubstituted 2-deoxystreptamines as next-generation aminoglycoside antibiotics.

UNLABELLED: The emerging epidemic of drug resistance places the development of efficacious and safe antibiotics in the spotlight of current research. Here, we report the design of next-generation aminoglycosides. Discovery efforts were driven by rational synthesis focusing on 4' alkylations of the aminoglycoside paromomycin, with the goal to alleviate the most severe and disabling side effect of aminoglycosides-irreversible hearing loss. Compounds were evaluated for target activity in in vitro ribosomal translation assays, antibacterial potency against selected pathogens, cytotoxicity against mammalian cells, and in vivo ototoxicity. The results of this study produced potent compounds with excellent selectivity at the ribosomal target, promising antibacterial activity, and little, if any, ototoxicity upon chronic administration. The favorable biocompatibility profile combined with the promising antibacterial activity emphasizes the potential of next-generation aminoglycosides in the treatment of infectious diseases without the risk of ototoxicity. IMPORTANCE: The ever-widening epidemic of multidrug-resistant infectious diseases and the paucity of novel antibacterial agents emerging from modern screening platforms mandate the reinvestigation of established drugs with an emphasis on improved biocompatibility and overcoming resistance mechanisms. Here, we describe the preparation and evaluation of derivatives of the established aminoglycoside antibiotic paromomycin that effectively remove its biggest deficiency, ototoxicity, and overcome certain bacterial resistance mechanisms.

Metformin protects against gentamicin-induced hair cell death in vitro but not ototoxicity in vivo.

Metformin (N,N-dimethylbiguanidine) is a widely employed oral hypoglycemic agent for the management of type 2 diabetes mellitus. Its antioxidant properties and safe clinical use raise the possibility of preventing gentamicin-induced hearing loss in patients. Therefore, we screened the usefulness of metformin against gentamicin toxicity in murine cochlear explants and in the guinea pig in vivo. We confirmed in organ culture that metformin blocks the gentamicin-induced translocation of endonuclease G into the nucleus of outer hair cells and attenuates hair cell loss. In vivo, gentamicin treatment with 80, 100, or 130mg/kg body weight for 14 days induced significant threshold shifts as determined by auditory brain stem responses. Metformin (30, 75, or 100mg/kg for 14 days) was well tolerated without any indication of auditory side effects. However, co-administration of metformin with gentamicin in various permutations did not prevent loss of auditory function. On the contrary, combined treatment at higher dosages aggravated the gentamicin-induced threshold shifts and caused additional adverse reactions including body weight loss and premature deaths in some animals. These results caution against the use of metformin co-treatment with aminoglycosides and confirm the need for in vivo studies in order to evaluate potentially protective agents selected by in vitro screens.

Designer aminoglycosides that selectively inhibit cytoplasmic rather than mitochondrial ribosomes show decreased ototoxicity: a strategy for the treatment of genetic diseases.

There is compelling evidence that aminoglycoside (AG) antibiotics can induce the mammalian ribosome to suppress disease-causing nonsense mutations and partially restore the expression of functional proteins. However, prolonged AG treatment can cause detrimental side effects in patients, including most prominently, ototoxicity. Recent mechanistic discussions have considered the relative contributions of mitochondrial and cytoplasmic protein synthesis inhibition to AG-induced ototoxicity. We show that AGs inhibit mitochondrial protein synthesis in mammalian cells and perturb cell respiration, leading to a time- and dose-dependent increase in superoxide overproduction and accumulation of free ferrous iron in mitochondria caused by oxidative damage of mitochondrial aconitase, ultimately leading to cell apoptosis via the Fenton reaction. These deleterious effects increase with the increased potency of AG to inhibit the mitochondrial rather than cytoplasmic protein synthesis, which in turn correlates with their ototoxic potential in both murine cochlear explants and the guinea pig in vivo. The deleterious effects of AGs were alleviated in synthetic derivatives specially designed for the treatment of genetic diseases caused by nonsense mutations and possessing low affinity toward mitochondrial ribosomes. This work highlights the benefit of a mechanism-based drug redesign strategy that can maximize the translational value of "readthrough therapy" while mitigating drug-induced side effects. This approach holds promise for patients suffering from genetic diseases caused by nonsense mutations.

Silencing near tRNA genes is nucleosome-mediated and distinct from boundary element function.

Transfer RNA (tRNA) genes and other RNA polymerase III transcription units are dispersed in high copy throughout nuclear genomes, and can antagonize RNA polymerase II transcription in their immediate chromosomal locus. Previous work in Saccharomyces cerevisiae found that this local silencing required subnuclear clustering of the tRNA genes near the nucleolus. Here we show that the silencing also requires nucleosome participation, though the nature of the nucleosome interaction appears distinct from other forms of transcriptional silencing. Analysis of an extensive library of histone amino acid substitutions finds a large number of residues that affect the silencing, both in the histone N-terminal tails and on the nucleosome disk surface. The residues on the disk surfaces involved are largely distinct from those affecting other regulatory phenomena. Consistent with the large number of histone residues affecting tgm silencing, survey of chromatin modification mutations shows that several enzymes known to affect nucleosome modification and positioning are also required. The enzymes include an Rpd3 deacetylase complex, Hos1 deacetylase, Glc7 phosphatase, and the RSC nucleosome remodeling activity, but not multiple other activities required for other silencing forms or boundary element function at tRNA gene loci. Models for communication between the tRNA gene transcription complexes and local chromatin are discussed.

Denture adhesive use in complete dentures: clinical recommendations and review of the literature.

This literature review sought to determine the advantages and disadvantages of denture adhesive use among complete denture patients. Manuscripts were obtained by searching the National Library of Medicine's PubMed database, Cochrane Collaboration Library, ADA Center for Evidence-Based Dentistry website, and EMBASE database. A total of 85 abstracts were reviewed, and 38 articles that met the inclusion criteria for this review were selected. The inclusion criteria included clinical trials and case series in which 10 or more patients were treated, as well as Cochrane collaboration reviews and in vitro studies where clinical relevance could be determined. The selected manuscripts were reviewed using a standardized manuscript review matrix. Although denture adhesives improve the retention and function of complete dentures, standardized guidelines are needed for the proper use, application, and removal of denture adhesives. Additionally, long-term studies are warranted on the biologic effects of denture adhesives. There is a need to establish a regular recall program for complete denture patients.

High-resolution x-ray structure and functional analysis of the murine norovirus 1 capsid protein protruding domain.

Murine noroviruses (MNV) are closely related to the human noroviruses (HuNoV), which cause the majority of nonbacterial gastroenteritis. Unlike HuNoV, MNV grow in culture and in a small-animal model that represents a tractable model to study norovirus biology. To begin a detailed investigation of molecular events that occur during norovirus binding to cells, the crystallographic structure of the murine norovirus 1 (MNV-1) capsid protein protruding (P) domain has been determined. Crystallization of the bacterially expressed protein yielded two different crystal forms (Protein Data Bank identifiers [PDB ID], 3LQ6 and 3LQE). Comparison of the structures indicated a large degree of structural mobility in loops on the surface of the P2 subdomain. Specifically, the A'-B' and E'-F' loops were found in open and closed conformations. These regions of high mobility include the known escape mutation site for the neutralizing antibody A6.2 and an attenuation mutation site, which arose after serial passaging in culture and led to a loss in lethality in STAT1(-/-) mice, respectively. Modeling of a Fab fragment and crystal structures of the P dimer into the cryoelectron microscopy three-dimensional (3D) image reconstruction of the A6.2/MNV-1 complex indicated that the closed conformation is most likely bound to the Fab fragment and that the antibody contact is localized to the A'-B' and E'-F' loops. Therefore, we hypothesize that these loop regions and the flexibility of the P domains play important roles during MNV-1 binding to the cell surface.