Giulia Paci

Debugging Eukaryotic Genetic Code Expansion for Site-Specific Click-PAINT Super-Resolution Microscopy.

Abstract Super‐resolution microscopy (SRM) greatly benefits from the ability to install small photostable fluorescent labels into proteins. Genetic code expansion (GCE) technology addresses this demand, allowing the introduction of small labeling sites, in the form of uniquely reactive noncanonical amino acids (ncAAs), at any residue in a target protein. However, low incorporation efficiency of ncAAs and high background fluorescence limit its current SRM applications. Redirecting the subcellular localization of the pyrrolysine‐based GCE system for click chemistry, combined with DNA‐PAINT microscopy, enables the visualization of even low‐abundance proteins inside mammalian cells. This approach links a versatile, biocompatible, and potentially unbleachable labeling method with residue‐specific precision. Moreover, our reengineered GCE system eliminates untargeted background fluorescence and substantially boosts the expression yield, which is of general interest for enhanced protein engineering in eukaryotes using GCE.

Labeling of virus components for advanced, quantitative imaging analyses

In recent years, investigation of virus–cell interactions has moved from ensemble measurements to imaging analyses at the single‐particle level. Advanced fluorescence microscopy techniques provide single‐molecule sensitivity and subdiffraction spatial resolution, allowing observation of subviral details and individual replication events to obtain detailed quantitative information. To exploit the full potential of these techniques, virologists need to employ novel labeling strategies, taking into account specific constraints imposed by viruses, as well as unique requirements of microscopic methods. Here, we compare strengths and limitations of various labeling methods, exemplify virological questions that were successfully addressed, and discuss challenges and future potential of novel approaches in virus imaging.

Fluorogenic Tetrazine-Siliconrhodamine Probe for the Labeling of Noncanonical Amino Acid Tagged Proteins

Tetrazine-bearing fluorescent labels enable site-specific tagging of proteins that are genetically manipulated with dienophile modified noncanonical amino acids. The inverse electron demand Diels-Alder reaction between the tetrazine and the dienophile fulfills the criteria of bioorthogonality allowing fluorescent labeling schemes of live cells. Here, we describe the detailed synthetic and labeling protocols of a near infrared emitting siliconrhodamine-tetrazine probe suitable for super-resolution imaging of residue-specifically engineered proteins in mammalian cells.

A Discipline-Enriched Dataset for Tracking the Computational Turn of European Universities

In recent years, academic research appears to have been going through a methodological turning point. The discussion around the impact that computational methods will have on traditional fields of study has been the focus of many calls for papers and panels at established conferences. However, despite the high prevalence of this topic in the academic debate, it remains very challenging to assess whether academia as a whole has been actually adopting more digital resources and methods during the recent years. We are currently studying this topic by combining hermeneutic and text mining practices while analyzing one of the primary research output of European universities, namely doctoral theses. In this work, we present an enriched dataset we created for addressing this research questions and the first results of the analyses we have conducted so far.