Simulation-based Reconstructed Diffusion unveils the effect of aging on protein diffusion in Escherichia coli.

We have developed Simulation-based Reconstructed Diffusion (SbRD) to determine diffusion coefficients corrected for confinement effects and for the bias introduced by two-dimensional models describing a three-dimensional motion. We validate the method on simulated diffusion data in three-dimensional cell-shaped compartments. We use SbRD, combined with a new cell detection method, to determine the diffusion coefficients of a set of native proteins in Escherichia coli. We observe slower diffusion at the cell poles than in the nucleoid region of exponentially growing cells, which is independent of the presence of polysomes. Furthermore, we show that the newly formed pole of dividing cells exhibits a faster diffusion than the old one. We hypothesize that the observed slowdown at the cell poles is caused by the accumulation of aggregated or damaged proteins, and that the effect is asymmetric due to cell aging.

SIMED-New Doc course, a matter of reflection.

BACKGROUND AND AIM: Obtaining a degree in medicine in Italy qualifies for the medical profession; this fact has entailed a newly qualified doctor's remarkable involvement on the medical activities of the National Health Service, especially during the Covid-19 pandemic. It is important to understand the knowledge of the newly qualified doctors and to create specific courses oriented to them. The aim of the study is to evaluate the impact of a peer learning course for the students who attend the last year of medicine school, with the purpose of defining the formal requests to integrate on the course. METHODS: A pre and post qualitative research has been carried out on SIMED-NEWDOC course. The course consisted on peer teaching lectures, as lecturers were resident doctors part of SIMED board. At the end of the course it has been submitted a survey to the participants, and data has been analyzed. RESULTS: The students enrolled were 139, the average of the participants was 27% of the registered. A qualitative evaluation questionnaire was submitted, the responses were 32 (86%). Average age was 25. Participants attending the last year of medicine school were 30 (95%). 40% of them declared to have attended at least 5 lessons. Among the course participants, 96% judged the course as very useful. CONCLUSIONS: All questionnaire results are useful to reflect on future projects. It is necessary to implement further educational projects to better understand the phenomenon, considering the positive impact that participants declared.

Super-resolving microscopy reveals the localizations and movement dynamics of stressosome proteins in Listeria monocytogenes.

The human pathogen Listeria monocytogenes can cope with severe environmental challenges, for which the high molecular weight stressosome complex acts as the sensing hub in a complicated signal transduction pathway. Here, we show the dynamics and functional roles of the stressosome protein RsbR1 and its paralogue, the blue-light receptor RsbL, using photo-activated localization microscopy combined with single-particle tracking and single-molecule displacement mapping and supported by physiological studies. In live cells, RsbR1 is present in multiple states: in protomers with RsbS, large clusters of stressosome complexes, and in connection with the plasma membrane via Prli42. RsbL diffuses freely in the cytoplasm but forms clusters upon exposure to light. The clustering of RsbL is independent of the presence of Prli42. Our work provides a comprehensive view of the spatial organization and intracellular dynamics of the stressosome proteins in L. monocytogenes, which paves the way towards uncovering the stress-sensing mechanism of this signal transduction pathway.

Protein diffusion in Escherichia coli cytoplasm scales with the mass of the complexes and is location dependent.

We analyze the structure of the cytoplasm by performing single-molecule displacement mapping on a diverse set of native cytoplasmic proteins in exponentially growing Escherichia coli. We evaluate the method for application in small compartments and find that confining effects of the cell membrane affect the diffusion maps. Our analysis reveals that protein diffusion at the poles is consistently slower than in the center of the cell, i.e., to an extent greater than the confining effect of the cell membrane. We also show that the diffusion coefficient scales with the mass of the used probes, taking into account the oligomeric state of the proteins, while parameters such as native protein abundance or the number of protein-protein interactions do not correlate with the mobility of the proteins. We argue that our data paint the prokaryotic cytoplasm as a compartment with subdomains in which the diffusion of macromolecules changes with the perceived viscosity.

Fluorescence-based sensing of the bioenergetic and physicochemical status of the cell.

Fluorescence-based sensors play a fundamental role in biological research. These sensors can be based on fluorescent proteins, fluorescent probes or they can be hybrid systems. The availability of a very large dataset of fluorescent molecules, both genetically encoded and synthetically produced, together with the structural insights on many sensing domains, allowed to rationally design a high variety of sensors, capable of monitoring both molecular and global changes in living cells or in in vitro systems. The advancements in the fluorescence-imaging field helped researchers to obtain a deeper understanding of how and where specific changes occur in a cell or in vitro by combining the readout of the fluorescent sensors with the spatial information provided by fluorescent microscopy techniques. In this review we give an overview of the state of the art in the field of fluorescent biosensors and fluorescence imaging techniques, and eventually guide the reader through the choice of the best combination of fluorescent tools and techniques to answer specific biological questions. We particularly focus on sensors for probing the bioenergetics and physicochemical status of the cell.

Influence of Fluorescent Protein Maturation on FRET Measurements in Living Cells.

Förster resonance energy transfer (FRET)-based sensors are a valuable tool to quantify cell biology, yet it remains necessary to identify and prevent potential artifacts in order to exploit their full potential. We show here that artifacts arising from slow donor mCerulean3 maturation can be substantially diminished by constitutive expression in both prokaryotic and eukaryotic cells, which can also be achieved by incorporation of faster-maturing FRET donors. We developed an improved version of the donor mTurquoise2 that matures faster than the parent protein. Our analysis shows that using equal maturing fluorophores in FRET-based sensors or using constitutive low expression conditions helps to reduce maturation-induced artifacts, without the need of additional noise-inducing spectral corrections. In general, we show that monitoring and controlling the maturation of fluorescent proteins in living cells is important and should be addressed in in vivo applications of genetically encoded FRET sensors.

Iron metallodrugs: stability, redox activity and toxicity against Artemia salina.

Iron metallodrugs comprise mineral supplements, anti-hypertensive agents and, more recently, magnetic nanomaterials, with both therapeutic and diagnostic roles. As biologically-active metal compounds, concern has been raised regarding the impact of these compounds when emitted to the environment and associated ecotoxicological effects for the fauna. In this work we assessed the relative stability of several iron compounds (supplements based on glucoheptonate, dextran or glycinate, as well as 3,5,5-trimethylhexanoyl (TMH) derivatives of ferrocene) against high affinity models of biological binding, calcein and aprotransferrin, via a fluorimetric method. Also, the redox-activity of each compound was determined in a physiologically relevant medium. Toxicity toward Artemia salina at different developmental stages was measured, as well as the amount of lipid peroxidation. Our results show that polymer-coated iron metallodrugs are stable, non-redox-active and non-toxic at the concentrations studied (up to 300 µM). However, TMH derivatives of ferrocene were less stable and more redox-active than the parent compound, and TMH-ferrocene displayed toxicity and lipid peroxidation to A. salina, unlike the other compounds. Our results indicate that iron metallodrugs based on polymer coating do not present direct toxicity at low levels of emission; however other iron species (eg. metallocenes), may be deleterious for aquatic organisms. We suggest that ecotoxicity depends more on metal speciation than on the total amount of metal present in the metallodrugs. Future studies with discarded metallodrugs should consider the chemical speciation of the metal present in the composition of the drug.