Evaluation of the bacterial ocular surface microbiome in clinically normal cats before and after treatment with topical erythromycin.

The ocular surface microbiome of veterinary species has not been thoroughly characterized using next generation sequencing. Furthermore, alterations in the feline ocular surface microbiome over time or following topical antibiotic treatment are unknown. Aims of this study were to further characterize the ocular surface microbiome of healthy cats and to identify whether there are microbial community changes over time and following topical antibiotic use. Twenty-four eyes from twelve adult, research-bred, female spayed domestic shorthaired cats were evaluated. Erythromycin ophthalmic ointment (0.5%) was applied to the ocular surface of one randomly assigned eye per cat three times daily for 7 days, while the fellow eye served as an untreated control. The ocular surface was sampled by swabbing the inferior conjunctival fornix of both eyes prior to initiating treatment (day 0), after 1 week of treatment (day 7), and 4 weeks after concluding treatment (day 35). Genomic DNA was extracted from the swabs and sequenced using primers that target the V4 region of bacterial 16S rRNA genes. At baseline, the most common bacterial phyla identified were Proteobacteria (42.4%), Firmicutes (30.0%), Actinobacteria (15.6%), and Bacteroidetes (8.1%). The most abundant bacterial families sequenced were Corynebacteriaceae (7.8%), Helicobacteraceae (7.5%), Moraxellaceae (6.1%), and Comamonadaceae (5.6%). Alpha and beta diversity measurements were largely unchanged in both treatment and control eyes over time. However, univariate and linear discriminant analyses revealed significant and similar changes in the abundance of some bacterial taxa over time in both treatment and control eyes. Overall, the feline ocular surface microbiome remained stable over time and following topical antibiotic therapy

Confirming the Revised C-Terminal Domain of the MscL Crystal Structure

The structure of the C-terminal domain of the mechanosensitive channel of large conductance (MscL) has generated significant controversy. As a result, several structures have been proposed for this region: the original crystal structure (1MSL) of the Mycobacterium tuberculosis homolog (Tb), a model of the Escherichia coli homolog, and, most recently, a revised crystal structure of Tb-MscL (2OAR). To understand which of these structures represents a physiological conformation, we measured the impact of mutations to the C-terminal domain on the thermal stability of Tb-MscL using circular dichroism and performed molecular dynamics simulations of the original and the revised crystal structures of Tb-MscL. Our results imply that this region is helical and adopts an α-helical bundle conformation similar to that observed in the E. coli MscL model and the revised Tb-MscL crystal structure

Defining the Role of the Tension Sensor in the Mechanosensitive Channel of Small Conductance

Mutations that alter the phenotypic behavior of the Escherichia coli mechanosensitive channel of small conductance (MscS) have been identified; however, most of these residues play critical roles in the transition between the closed and open states of the channel and are not directly involved in lipid interactions that transduce the tension response. In this study, we use molecular dynamic simulations to predict critical lipid interacting residues in the closed state of MscS. The physiological role of these residues was then investigated by performing osmotic downshock assays on MscS mutants where the lipid interacting residues were mutated to alanine. These experiments identified seven residues in the first and second transmembrane helices as lipid-sensing residues. The majority of these residues are hydrophobic amino acids located near the extracellular interface of the membrane. All of these residues interact strongly with the lipid bilayer in the closed state of MscS, but do not face the bilayer directly in structures associated with the open and desensitized states of the channel. Thus, the position of these residues relative to the lipid membrane appears related to the ability of the channel to sense tension in its different physiological states

Detection and identification of genetic material via single-molecule conductance

The ongoing discoveries of RNA modalities (for example, non-coding, micro and enhancer) have resulted in an increased desire for detecting, sequencing and identifying RNA segments for applications in food safety, water and environmental protection, plant and animal pathology, clinical diagnosis and research, and bio-security. Here, we demonstrate that single-molecule conductance techniques can be used to extract biologically relevant information from short RNA oligonucleotides, that these measurements are sensitive to attomolar target concentrations, that they are capable of being multiplexed, and that they can detect targets of interest in the presence of other, possibly interfering, RNA sequences. We also demonstrate that the charge transport properties of RNA:DNA hybrids are sensitive to single-nucleotide polymorphisms, thus enabling differentiation between specific serotypes of Escherichia coli. Using a combination of spectroscopic and computational approaches, we determine that the conductance sensitivity primarily arises from the effects that the mutations have on the conformational structure of the molecules, rather than from the direct chemical substitutions. We believe that this approach can be further developed to make an electrically based sensor for diagnostic purposes