Circadian clocks are intracellular systems that orchestrate metabolic processes in anticipation of sunrise and sunset by providing an internal representation of local time. Because the ~24-h metabolic rhythms they produce are important to health across diverse life forms there is growing interest in their mechanisms. However, mechanistic studies are challenging in vivo due to the complex, that is, poorly defined, milieu of live cells. Recently, we reconstituted the intact circadian clock of cyanobacteria in vitro. It oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of individual clock proteins and promoter DNA simultaneously under defined conditions without user intervention. We found that reproducibility of the reactions required strict adherence to the quality of each recombinant clock protein purified from Escherichia coli. Here, we provide protocols for preparing in vitro clock samples so that other labs can ask questions about how changing environments, like temperature, metabolites, and protein levels are reflected in the core oscillator and propagated to regulation of transcription, providing deeper mechanistic insights into clock biology.

Polymeric barrier membranes are used in periodontal applications to prevent fibroblastic cell migration into the cavities of bone tissue and to properly guide the proliferation of tissues. In this study, the fabrication, characterization, bioactivity, and in vitro biological properties of polyvinyl alcohol-based nanofibrous membranes containing nano-sized 45S5 bioactive glass (BG) loaded with chlorhexidine (CH) gluconate with biocompatible, bioactive, and antibacterial properties for using as dental barrier membranes were investigated. Nanofibrous membranes with an average fiber diameter, pore size, and porosity of 210 nm, 24.73 μm, and 12.42%, respectively, were loaded with 1% and 2% CH, and the release profile was investigated. The presence of BG in the membranes promoted fibroblastic proliferation and the presence of CH provided antibacterial properties. Nanofibrous membranes exhibit a high ability to restrict bacterial growth while fulfilling the necessary conditions for use as a dental barrier thanks to their low swelling rates, significant surface bioactivities, and appropriate degradation levels.