Identifying Ciliary Proteins in Mammalian Retinas using a Gentle Extraction Method.

Mutations in retinal primary cilia are responsible for human blindness but the mechanisms are not fully understood (Wheway et al., 2014). Characterizing the proteome of an organelle such as cilia, is a fruitful way to understand its function but methods often require considerable sample quantities. Here we develop a method to isolate the primary cilia of photoreceptor cells from bovine retinas. Through LC/MS/MS proteomics analysis we identify proteins enriched for cilia function including ciliopathy disease. This study shows our method can be used to isolate retinal primary cilia to obtain sufficient quantities of native protein samples.

Scaffold Matcher: A CMA-ES based algorithm for identifying hotspot aligned peptidomimetic scaffolds.

The design of protein interaction inhibitors is a promising approach to address aberrant protein interactions that cause disease. One strategy in designing inhibitors is to use peptidomimetic scaffolds that mimic the natural interaction interface. A central challenge in using peptidomimetics as protein interaction inhibitors, however, is determining how best the molecular scaffold aligns to the residues of the interface it is attempting to mimic. Here we present the Scaffold Matcher algorithm that aligns a given molecular scaffold onto hotspot residues from a protein interaction interface. To optimize the degrees of freedom of the molecular scaffold we implement the covariance matrix adaptation evolution strategy (CMA-ES), a state-of-the-art derivative-free optimization algorithm in Rosetta. To evaluate the performance of the CMA-ES, we used 26 peptides from the FlexPepDock Benchmark and compared with three other algorithms in Rosetta, specifically, Rosetta's default minimizer, a Monte Carlo protocol of small backbone perturbations, and a Genetic algorithm. We test the algorithms' performance on their ability to align a molecular scaffold to a series of hotspot residues (i.e., constraints) along native peptides. Of the 4 methods, CMA-ES was able to find the lowest energy conformation for all 26 benchmark peptides. Additionally, as a proof of concept, we apply the Scaffold Match algorithm with CMA-ES to align a peptidomimetic oligooxopiperazine scaffold to the hotspot residues of the substrate of the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our implementation of CMA-ES into Rosetta allows for an alternative optimization method to be used on macromolecular modeling problems with rough energy landscapes. Finally, our Scaffold Matcher algorithm allows for the identification of initial conformations of interaction inhibitors that can be further designed and optimized as high-affinity reagents.

Validating cleanability of dental rotary diamond instruments soiled with 2 clinically relevant dental test soil components.

BACKGROUND: The aim of this study was to produce a dental test soil, with 2 clinically relevant soil components, to be quantified for cleaning process validation. Another goal was to soil diamond instruments with the 2 soil components and validate the efficacy of cleaning instructions, developed and detailed in this study, using both qualitative and quantitative techniques. METHODS: To simulate worst-case clinical use conditions, the authors used each soiled instrument to prepare a 9-millimeter-deep access cavity on a noncarious extracted molar. Afterward, the authors applied a mixture of pooled human saliva and blood test soil to each instrument and air-dried it for 30 minutes. The authors cleaned each instrument using documented multistep cleaning instructions, which were then validated via both qualitative and quantitative assessment of protein and enamel-dentin residues using spectrophotometric analysis and microscopy images. RESULTS: After thorough cleaning, neither protein nor enamel-dentin residues were found at quantifiable levels (spectrophotometric analysis) on the soiled and cleaned diamond instruments, which was qualitatively verified (microscopy images). CONCLUSIONS: The results of this study show the successful development of a dental test soil with 2 clinically relevant soil components. Furthermore, using these soil components as test markers, the authors found that when the established cleaning instructions are properly followed, a soiled diamond instrument can be cleaned in a quantifiable manner. PRACTICAL IMPLICATIONS: Thorough cleaning is a critical step in reprocessing multiuse dental instruments. In accordance with US Food and Drug Administration guidance, the described process for quantification of soil components, using 2 clinically relevant soil markers, on cleaned diamond instruments can be helpful to dental instrument manufacturers in the development and validation of cleaning instructions for their reusable instruments.