Biology subjects

Fantini, M.

Publications and source records attributed to Fantini, M..

5 recordsLinked to original sources

bioRxiv2026

Semi-permeable capsules enable parallel cultivation and live microscopic observations of microbial eukaryotes

Semi-permeable capsules (SPCs) create enclosed porous microenvironments, diffusible to only small proteins and macromolecules. This presents a powerful tool for single cell observation, isolation, and sequencing. However, their range of use for sustaining viable microbial eukaryotes is largely unexplored. Single-cell eukaryotes are often understudied, with a wealth of unknown lifecycles, culturing methods and inter-microbial interactions, which are difficult to visualize. Here, we show that eukaryotes from eight different supergroups can be captured and propagated in SPCs. Encapsulation allowed observations of cell stages, motility and growth in a traceable and parallelized manner.

microbiology↗

bioRxiv2021

Aggregation is a context-dependent constraint on protein evolution

Solubility is a requirement for many cellular processes. Loss of solubility and aggregation can lead to the partial or complete abrogation of protein function. Thus, understanding the relationship between protein evolution and aggregation is an important goal. Here, we analysed two deep mutational scanning experiments to investigate the role of protein aggregation in molecular evolution. In one data set, mutants of a protein involved in RNA biogenesis and processing, human TAR DNA binding protein 43 (TDP-43), were expressed in S. cerevisiae. In the other data set, mutants of a bacterial enzyme that controls resistance to penicillins and cephalosporins, TEM-1 beta-lactamase, were expressed in E. coli under the selective pressure of an antibiotic treatment. We found that aggregation differentiates the effects of mutations in the two different cellular contexts. Specifically, aggregation was found to be associated with increased cell fitness in the case of TDP-43 mutations, as it protects the host from aberrant interactions. By contrast, in the case of TEM-1 beta-lactamase mutations, aggregation is linked to a decreased cell fitness due to inactivation of protein function. Our study shows that aggregation is an important context-dependent constraint of molecular evolution and opens up new avenues to investigate the role of aggregation in different cellular contexts.

bioinformatics↗

bioRxiv2019

Protein structure without structure determination: direct coupling analysis based on in vitro evolution

Protein structure is tightly inter-twined with function according to the laws of evolution. Understanding how structure determines function has been the aim of structural biology for decades. Here, we have wondered instead whether it is possible to exploit the function for which a protein was evolutionary selected to gain information on protein structure and on the landscape explored during the early stages of molecular and natural evolution. To answer to this question, we developed a new methodology, which we named CAMELS (Coupling Analysis by Molecular Evolution Library Sequencing), that is able to obtain the in vitro evolution of a protein from an artificial selection based on function. We were able to observe with CAMELS many features of the TEM-1 beta lactamase local fold exclusively by generating and sequencing large libraries of mutational variants. We demonstrated that we can, whenever a functional phenotypic selection of a protein is available, sketch the structural and evolutionary landscape of a protein without utilizing purified proteins, collecting physical measurements or relying on the pool of natural protein variants.

evolutionary biology↗

bioRxiv2017

New Techniques for Ancient Proteins: Direct Coupling Analysis Applied on Proteins involved in Iron Sulfur Cluster Biogenesis

Direct coupling analysis (DCA) is a powerful tool based on protein evolution and introduced to predict protein fold and protein-protein interactions which has been applied also to the prediction of entire interactomes. We have used DCA to analyse three proteins of the iron-sulfur biogenesis machine, an essential metabolic pathway conserved in all organisms. We show that, although based on a relatively small number of sequences due to its distribution in genomes, we can correctly recapitulate all the features of the fold of the CyaY/frataxin family, a protein involved in the human disease Friedreichs ataxia. This result gave us confidence in the use of this tool. Application of DCA to the iron-sulfur cluster scaffold protein IscU, which has been suggested to function both as an ordered and a disordered form, allows us to clearly distinguish evolutionary traces of the structured species, suggesting that, if present in the cell, the disordered form has not left any evolutionary imprinting. We observe instead, for the first time, direct indications of how the protein can dimerize head-to-head and bind 4Fe4S clusters. Analysis of the alternative scaffold protein IscA provides strong support to a coordination of the cluster mediated by a dimeric rather than a tetrameric form as previously suggested. Our analysis also suggests the presence in solution of a mixture of monomeric and dimeric species and guide us to the prevalent one. Finally, we used DCA to analyse protein-protein interactions between some of these proteins and discuss the potentialities and the limitations of the method.

bioinformatics↗

bioRxiv2016

Assessment of Antibody Library Diversity through Next Generation Sequencing and Technical Error Compensation.

Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its diversity, i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library diversity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Diversity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring diversity from such a small sample is, however, very rudimental and gives limited information about the real complexity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library diversity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the diversity, taking in consideration the sequencing error.

bioinformatics↗