Biocat

Abstract Since its burst in early 2020, the Covid-19 pandemic has deeply affected every aspect of daily life, from international trade and travelling to restrictions on an individual level, becoming a complex multi-level and highly multi-faceted problem. Due to its overarching influence and deep impact, it can be seen as one of the most disruptive Grand Challenges of our time. Different from most other lasting Grand Challenges, such as Climate Change, the pandemic exerted its influence with little ramp-up, rapidly transforming health and health systems, human lives, goods and economic flows, decision-making mechanisms, research and innovation, and many other aspects of life in a very short span of time.Grand Challenges require extraordinary efforts from society as a whole since they need holistic, effective, collaborative endeavours to solve them. One such unique orchestrated effort can be observed in the subsequent series of virtual massive EUvsVirus (https://www.euvsvirus.org/) events and committed collaborations (‘hackathon’, ‘matchathon’, ‘launchathon’, ‘community’, ‘EIC Covid platform’, and the unparalleled ‘Academia Diffusion Experiment’ [ADE], analysed in chapter “Academia Diffusion Experiment: Trailblazing the Emergence from Co-Creation” of this book).While this chapter explains ‘what’ has been produced with the ADE, inspired by the EUvsVirus phenomenon, the ADE chapter describes ‘how’ it has been done. Both are extremely unique in terms of content, procedure, motivation, collaboration, effects—and they attempt to trailblaze at highest level co-creation, co-evolution, and co-dreaming. Hence, situated as the last chapters of this book.This chapter will shed light on the EUvsVirus events, where over 30,000 individuals from 40 countries came together and addressed the complexity of this massive challenge in a pioneering and groundbreaking way. The chapter is focused on analysing the EUvsVirus hackathon (alongside its mentioned unique spillovers) as a tool, method, and process capable of channelling and activating individuals’ and institutions’ concerns, wills, and commitments into a unique orchestrated open, collaborative response to an urgent Grand Challenge, the pandemic. We are producing a multi-vortex tornado model, resembling the EUvsVirus phenomenon, its components, mechanisms, behaviour and how to replicate it to achieve such disruptive, global organisational effort of co-creation. Especially, the emergence of such collaboration in the face of such urgency leads to the assumption that there are crucial lessons to be learned from this endeavour, quite fittingly encapsulated by these words:‘We are learningThat though we weren't ready for this,We have been readied by it’.Amanda Gorman’s New Year poem (https://amandagormanbooks.com/#the-hill-we-climb-and-other-poems or https://edition.cnn.com/videos/tv/2022/01/06/exp-amanda-gorman-nye-poem.cnn)

Inspired by EUvsVirus, this chapter focuses on the Academia Diffusion Experiment (ADE) with more than 40 scholars and practitioners around the globe, who transform practice into science through co-creating, co-evolving, and co-dreaming. The chapter first introduces EUvsVirus as the context of the ADE and then uncovers the Denica 2.0 managerial method deployed to facilitate it, its primary constituents, governing principles, participants, information, and risk management. Finally, the chapter reflects the learnings from the ADE by discussing its potentials, problems, and challenges.

As a result of exome-based sequencing work performed by the DDD study, de novo variants in CNOT3 have emerged as a newly recognised cause of a developmental disorder. This paper describes molecular and clinical details of 16 probands with developmental disorders and de novo CNOT3 variants. It is the first such description of the developmental phenotype associated with CNOT3 variants. Eight of these cases were discovered as part of the DDD study, while the other eight were found as a result of large-scale sequencing work performed by other groups. A highly specific phenotype was not recognised in these 16 cases. The most consistent phenotypic features seen in subjects with de novo variants in CNOT3 were hypotonia, relatively small stature, developmental delay, behavioural problems and intellectual disability. There is no easily recognisable facial phenotype, but some common dysmorphic features such as anteverted nares, thin upper lip and low set eyebrows were shared among some of the probands. Haploinsufficiency appears to be the most likely mechanism of action, with eight cases found to have protein-truncating variants. Of the other eight cases (all missense variants), three share an amino acid substitution at the same position which may therefore represent an important functional domain.

The endo-polygalacturonase enzyme (endoPG: EC 3.2.1.15) plays an important role in the fruit juice and wine industries, so the development of new tools for the quantitative and qualitative analysis of its enzymatic action is necessary. In this work, we report the development of a simple, fast and practical method that did not use any chemical reagent to identify and evaluate the action of the endoPG enzyme, produced by the yeast Kluyveromyces marxianus CCT3172, using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy combined with principal component analysis-linear discriminant analysis (PCA-LDA). This method evaluated the action of the endoPG enzyme on the polygalacturonic acid (PGA) substrate at 5 different times (0, 10, 15, 20 and 30 minutes), and at each time interval the samples were analyzed by ATR-FTIR. It was demonstrated that there was clear segregation between the samples that were and that were not subjected to the action of the endoPG enzyme, and it was also possible to distinguish the samples that were subjected to different incubation times with the enzyme. Through PCA-LDA it was possible to obtain wavelengths that are biomarkers for this enzymatic reaction and the observed changes as a function of hydrolysis duration were found to be in agreement with the breakdown of the glycosidic chain (1011 cm-1-CH-O- CH stretching) of PGA and release of oligosaccharides (1078 cm-1 C-OH elongation). The activity of the endoPG enzyme and the release of galacturonic acid were verified by the dinitrosalicylic acid (DNS) method in all samples. The efficacy of an automatic classifier using a principal component analysis-linear discriminant classifier (PCA-LDC) was evaluated to diagnose the action of the endoPG enzyme. The results showed an accuracy of 100% for the identification of the endoPG enzyme action and from 91.67% to 100% for classification according to the hydrolysis duration in which PGA was exposed to endoPG. The present study indicates that this methodology may be a new approach for the qualitative evaluation of the endoPG enzyme with the potential to be used in laboratories and industries.

Novozym 435 (N435) is a commercially available immobilized lipase produced by Novozymes with its advantages and drawbacks.

Structure-guided reshaping the substrate-binding pocket of ameso-diaminopimelate dehydrogenase (StDAPDH) led to a mutant W121L/H227I, which catalyzed the enantioselective reductive amination of some sterically bulky 2-keto acids.

Molecular dynamics (MD) simulations, in combination with the Markov-state model (MSM), were applied to probe CO2 diffusion from an aqueous solution into the active site of human carbonic anhydrase II (hCA-II), an enzyme useful for enhanced CO2 capture and utilization. The diffusion process in the hydrophobic pocket of hCA-II was illustrated in terms of a two-dimensional free-energy landscape. We found that CO2 diffusion in hCA-II is a rate-limiting step in the CO2 diffusion-binding-reaction process. The equilibrium distribution of CO2 shows its preferential accumulation within a hydrophobic domain in the protein core region. An analysis of the committors and reactive fluxes indicates that the main pathway for CO2 diffusion into the active site of hCA-II is through a binding pocket where residue Gln136 contributes to the maximal flux. The simulation results offer a new perspective on the CO2 hydration kinetics and useful insights toward the development of novel biochemical processes for more efficient CO2 sequestration and utilization.

The proline analogue (2S,3aS,7aS)-octahydroindole-2-carboxylic acid (Oic) has been previously applied as a proline substitute in pharmocologically active peptides and as a structural component of the antihypertensive drug Perindopril. Herein, we describe the formation of an oligoproline structure by an Oic oligomer. A series of Oic oligomers were investigated to show the structural and energetic contribution of appended residues to the structure. NMR investigation of these oligomers revealed an all-trans amide bond structure, and we provide evidence that a cascade-like mechanism is responsible for the all-trans folding cooperativity. X-ray crystallography of the Oic-hexapeptide clearly demonstrates that the all-trans structure of the Oic oligomer is a polyproline II helix. Thus, as a hydrophobic proline analog with a highly stable trans-amide bond, Oic represents an ideal building block for hydrophobic sites of polyproline II structures in biologically relevant contexts.

This study focuses on the expansion of the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) towards the l-enantiomers of racemic styrylalanines rac-1a-d - which are less studied and synthetically challenging unnatural amino acids - by reshaping the aromatic binding pocket of the active site of PcPAL by point mutations. Ammonia elimination from l-styrylalanine (l-1a) catalyzed by non-mutated PcPAL (wt-PcPAL) took place with a 777-fold lower kcat/KM value than the deamination of the natural substrate, l-Phe. Computer modeling of the reactions catalyzed by wt-PcPAL indicated an unproductive and two major catalytically active conformations and detrimental interactions between the aromatic moiety of l-styrylalanine, l-1a, and the phenyl ring of the residue F137 in the aromatic binding region of the active site. Replacing the residue F137 by smaller hydrophobic residues resulted in a small mutant library (F137X-PcPAL, X being V, A, and G), from which F137V-PcPAL could transform l-styrylalanine with comparable activity to that of the wt-PcPAL with l-Phe. Furthermore, F137V-PcPAL showed superior catalytic efficiency in the ammonia elimination reaction of several racemic styrylalanine derivatives (rac-1a-d) providing access to d-1a-d by kinetic resolution, even though the d-enantiomers proved to be reversible inhibitors. The enhanced catalytic efficiency of F137V-PcPAL towards racemic styrylalanines rac-1a-d could be rationalized by molecular modeling, indicating the more relaxed enzyme-substrate complexes and the promotion of conformations with higher catalytic activities as the main reasons. Unfortunately, ammonia addition onto the corresponding styrylacrylates 2a-d failed with both wt-PcPAL and F137V-PcPAL. The low equilibrium constant of the ammonia addition, the poor ligand binding affinities of 2a-d, and the non-productive binding states of the unsaturated ligands 2a-d within the active sites of either wt-PcPAL or F137V-PcPAL - as indicated by molecular modeling - might be responsible for the inactivity of the PcPAL variants in the reverse reaction. Modeling predicted that the F137V mutation is beneficial for the KRs of 4-fluoro-, 4-cyano- and 4-bromostyrylalanines, but non-effective for the KR process of 4-trifluoromethylstyrylalanine.

This review on the biochemistry of W-containing enzymes, or tungstoenzymes, starts and ends with the question: “why tungsten?” Structural, kinetic and mechanistic properties of the enzymes are evaluated and related to the common division into two families and several classes. Experimental inconsistencies are identified and proposed models are questioned as a way to determine what we now know and what we still should find out in order to be able to address the “why tungsten?” question in sufficient (bio)chemical depth. Specific recommendations are made to re-direct research on tungstoenzymes in order to cover key dark spots in our present knowledge.