Elucidation and control of low and high active populations of alkaline phosphatase molecules for quantitative digital bioassay

Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP-based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single-molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP-based quantitative digital bioassays. This study aims quantitative analysis of single-molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half-active and fully active portions. Digital assays with serial buffer exchange uncovered single-molecule Michaelis-Menten kinetics of ALP; half-active molecules have halved values of the catalytic turnover rate, kcat, and the rate constant of productive binding, kon, of the fully active molecules. These findings suggest that half-active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell-free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, revealing that inactive monomer units of ALP are deficient in disulfide bond formation and zinc ion coordination, and also offering the way to prepare homogenous and active populations of ALP for quantitative digital bioassays of ALP.

The System Thinking Perspective in the Open-Innovation Research: A Systematic Review

The new logics of competitions are mostly based on exploiting relationships to implement new mechanisms in managing Knowledge. Today, a successful company should be, lean, modular, and with a smart approach to new products development. In this context, the source of competitive advantage cannot be found into a static heterogeneity of resources, but companies must be able to create and manage a dynamic competitive process to continuously reinvent their products/services and to re-combine their resources with their partners’ ones. A paradigm for this behavior is the Open Innovation one, as created by Chesbrough. According to the rules of this paradigm, companies have to acknowledge that they operate in a network of relationships, they must be open to cooperate with their external partners, and they must not try to limit their actions in reaching only for some pre-defined result. So, Open Innovation Networks appear to be similar to those described by the scholars in the Complex Adaptive Systems field where the actions of the system, and of its parts, are the result of the various actors’ interactions in an emergent way. In this paper, we use a Systematic Literature Review approach to explore how the main topics in the System Thinking Perspective, and in particular, those related to Complex Systems, are linked to the Open Innovation studies.