Integrated high-content quantification of intracellular ROS levels and mitochondrial morphofunction

Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-contentmicroscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow, and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells

The Fuzziness in Molecular, Supramolecular, and Systems Chemistry

Fuzzy Logic is a good model for the human ability to compute words. It is based on the theory of fuzzy set. A fuzzy set is different from a classical set because it breaks the Law of the Excluded Middle. In fact, an item may belong to a fuzzy set and its complement at the same time and with the same or different degree of membership. The degree of membership of an item in a fuzzy set can be any real number included between 0 and 1. This property enables us to deal with all those statements of which truths are a matter of degree. Fuzzy logic plays a relevant role in the field of Artificial Intelligence because it enables decision-making in complex situations, where there are many intertwined variables involved. Traditionally, fuzzy logic is implemented through software on a computer or, even better, through analog electronic circuits. Recently, the idea of using molecules and chemical reactions to process fuzzy logic has been promoted. In fact, the molecular word is fuzzy in its essence. The overlapping of quantum states, on the one hand, and the conformational heterogeneity of large molecules, on the other, enable context-specific functions to emerge in response to changing environmental conditions. Moreover, analog input–output relationships, involving not only electrical but also other physical and chemical variables can be exploited to build fuzzy logic systems. The development of “fuzzy chemical systems” is tracing a new path in the field of artificial intelligence. This new path shows that artificially intelligent systems can be implemented not only through software and electronic circuits but also through solutions of properly chosen chemical compounds. The design of chemical artificial intelligent systems and chemical robots promises to have a significant impact on science, medicine, economy, security, and wellbeing. Therefore, it is my great pleasure to announce a Special Issue of Molecules entitled “The Fuzziness in Molecular, Supramolecular, and Systems Chemistry.” All researchers who experience the Fuzziness of the molecular world or use Fuzzy logic to understand Chemical Complex Systems will be interested in this book

Multifunctional RNA-binding proteins influence mRNA abundance and translational efficiency of distinct sets of target genes

RNA-binding proteins (RBPs) can regulate more than a single aspect of RNA metabolism. We searched for such previously undiscovered multifunctionality within a set of 143 RBPs, by defining the predictive value of RBP abundance for the transcription and translation levels of known RBP target genes across 80 human hearts. This led us to newly associate 27 RBPs with cardiac translational regulation in vivo. Of these, 21 impacted both RNA expression and translation, albeit for virtually independent sets of target genes. We highlight a subset of these, including G3BP1, PUM1, UCHL5, and DDX3X, where dual regulation is achieved through differential affinity for target length, by which separate biological processes are controlled. Like the RNA helicase DDX3X, the known splicing factors EFTUD2 and PRPF8 - all identified as multifunctional RBPs by our analysis - selectively influence target translation rates depending on 5' UTR structure. Our analyses identify dozens of RBPs as being multifunctional and pinpoint potential novel regulators of translation, postulating unanticipated complexity of protein-RNA interactions at consecutive stages of gene expression

Understanding the relationship between histone acetylation and bromodomain targeting

DNA exists in a compact structure, known as chromatin. Chromatin enables a cell to compact its DNA into an individual cell, however, it limits the ability for regulatory proteins to access the underlying DNA. The cell has devised mechanisms to overcome this, including the covalent modification of histone tails and the use of ATP-dependent chromatin remodelers. The bromodomain-acetyl (BD-Ac) interaction is one of the most widespread interactions that links covalent modifications to a biological response. The work in this dissertation focuses on gaining a better understanding of how multiple BDs engage acetylation, how loss of the BD module can alter chromatin structure, and lastly, how the BD-Ac interaction can be targeted for cancer therapeutics. We focused on a subunit of the PBAF chromatin remodeling subunit, PBRM1, that is frequently mutated in clear cell renal cell carcinoma. This enabled us to simultaneously assess how a BD interacts with chromatin and the effect of mutating BDs on chromatin interactions, while gaining insight into how PBRM1 loss may be driving tumorigenesis. We show that PBRM1 BD2 and BD4 are the primary BDs responsible for mediating interactions with transcriptionally active regions of the genome through binding to H3K14ac and H3K4me3. The neighboring BDs enhance (BD1, BD5) or inhibit (BD3) binding of these primary BDs, while mutations within these BDs attenuate PBRM1 chromatin interactions. Loss of PBRM1 alters chromatin organization and enhancer maintenance, resulting in altered gene expression. Because the BD-Ac interaction is frequently disrupted in cancers, inhibitors of this interaction were developed. In the last part of this work we examine the genome-wide effect of histone deacetylase inhibitors (HDACi) on H4 acetylation (H4ac) and targeting of the BD-containing protein, BRD4. HDACi preferentially target regions of the genome with preexisting acetylation, most notably gene bodies. Highly transcribed genes were most affected by HDACi, with increased H4ac and BRD4 binding in the gene bodies. Together these findings will help future studies better understand how multiple reader domains engage chromatin and how disruption of this interaction can both promote cancer and be used as a therapeutic target.Doctor of Philosoph

Structure and dynamics of core-periphery networks

Recent studies uncovered important core/periphery network structures characterizing complex sets of cooperative and competitive interactions between network nodes, be they proteins, cells, species or humans. Better characterization of the structure, dynamics and function of core/periphery networks is a key step of our understanding cellular functions, species adaptation, social and market changes. Here we summarize the current knowledge of the structure and dynamics of "traditional" core/periphery networks, rich-clubs, nested, bow-tie and onion networks. Comparing core/periphery structures with network modules, we discriminate between global and local cores. The core/periphery network organization lies in the middle of several extreme properties, such as random/condensed structures, clique/star configurations, network symmetry/asymmetry, network assortativity/disassortativity, as well as network hierarchy/anti-hierarchy. These properties of high complexity together with the large degeneracy of core pathways ensuring cooperation and providing multiple options of network flow re-channelling greatly contribute to the high robustness of complex systems. Core processes enable a coordinated response to various stimuli, decrease noise, and evolve slowly. The integrative function of network cores is an important step in the development of a large variety of complex organisms and organizations. In addition to these important features and several decades of research interest, studies on core/periphery networks still have a number of unexplored areas.Comment: a comprehensive review of 41 pages, 2 figures, 1 table and 182 reference