Quantitative Biology

Micromanipulations, perfusions and measurements performed using glass microelectrodes filled with an electrolyte is a conventional technique for experimental morphological and membrane electrophysiological studies at a single cell and membrane surface level. The typical (effective) diameter of the end of the glass microelectrode is from 500 up to less than 100 nm, which prevents one from observing it using a standard optical microscope in accordance with the optical resolution criteria, since the diameter less than 500 nm is indistinguishable within the interference zone. Microprocessor programming of the puller (microforge) that provides pulling and tearing allows to obtain in certain regimes the adjusted diameter and shape of the micropipette tip, although this result is not fully controlled due to the above limitations. In this connection it is necessary to design the control devices for the micropipette tips both at the preparation and operation stages (intracellular or extracellular insertion). This method also should provide visualization of the processes occurring upon interaction of the microelectrode tip with the cell in real time, depending on the electrode type and state, which allows to level the artifacts arising with the systematic error frequency from the uncontrolled operation of the micropipette tip after different ways of the microelectrode filling with the electrolyte. We propose an installation scheme that solves the above problems by means of introducing an interferometric device for microscopic control of the microelectrode and micromanipulator or microperfusor, for the first time for a given type of optical instruments combined with the interferometric optical scheme.

Multiple Sclerosis (MS) is a disorder that usually appears in adults in their thirties. It has a prevalence that ranges between 2 and 150 per 100 000. Epidemiological studies of MS have provided hints on possible causes for the disease ranging from genetic, environmental and infectious factors to other factors of vascular origin. Despite the tremendous effort spent in the last few years, none of the hypotheses formulated so far has gained wide acceptance and the causes of the disease remain unknown. From a clinical point of view, a high correlation has been recently observed between MS and Chronic Cerebro-Spinal Venous Insufficiency (CCSVI) in a statistically significant number of patients. In this pathological situation CCSVI may induce alterations of blood pressure in brain microvessels, thereby perturbing the exchange of small hydrophilic molecules between the blood and the external cells. In the presence of large pressure alterations it cannot be excluded also the leakage of macromolecules that otherwise would not cross the vessel wall. All these disorders may trigger immune defenses with the destruction of myelin as a side effect. In the present work we investigate the role of perturbed blood pressure in brain microvessels as driving force for an altered exchange of small hydrophilic solutes and leakage of macromolecules into the interstitial fluid. With a simplified, yet realistic, model we obtain closed-form steady-state solutions for fluid flow and solute transport across the microvessel wall. Finally, we use these results (i) to interpret experimental data available in the literature and (ii) to carry out a preliminary analysis of the disorder in the exchange processes triggered by an increase of blood pressure, thereby relating our preliminary results to the hypothesised vascular connection to MS.

Pyroptosis is an inflammatory mode of cell death that can contribute to the cytokine storm associated with severe cases of coronavirus disease 2019 (COVID-19). The formation of the NLRP3 inflammasome is central to pyroptosis, which may be induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Inflammasome formation, and by extension pyroptosis, may be inhibited by certain anti-inflammatory drugs. In this study, we present a single-cell mathematical model that captures the formation of the NLRP3 inflammasome, pyroptotic cell death and responses to anti-inflammatory intervention that hinder the formation of the NLRP3 inflammasome. The model is formulated in terms of a system of ordinary differential equations (ODEs) that describe the dynamics of the biological components involved in pyroptosis. Our results demonstrate that an anti-inflammatory drug can delay the formation of the NLRP3 inflammasome, and thus may alter the mode of cell death from inflammatory (pyroptosis) to non-inflammatory e.g., apoptosis). The single-cell model is being implemented in a SARS-CoV-2 Tissue Simulator, in collaboration with a multidisciplinary coalition investigating within host-dynamics of COVID-19. In this paper, we provide an overview of the SARS-CoV-2 Tissue Simulator and highlight the effects of pyroptosis on a cellular level.

Macromolecular crowding in living biological cells effects subdiffusion of larger biomolecules such as proteins and enzymes. Mimicking this subdiffusion in terms of random walks on a critical percolation cluster, we here present a case study of EcoRV restriction enzymes involved in vital cellular defence. We show that due to its so far elusive propensity to an inactive state the enzyme avoids non-specific binding and remains well-distributed in the bulk cytoplasm of the cell. Despite the reduced volume exploration capability of subdiffusion processes, this mechanism guarantees a high efficiency of the enzyme. By variation of the non-specific binding constant and the bond occupation probability on the percolation network, we demonstrate that reduced non-specific binding are beneficial for efficient subdiffusive enzyme activity even in relatively small bacteria cells. Our results corroborate a more local picture of cellular regulation.

We examine immunostaining experimental data for the formation of the strip 2 of even−skipped ( eve ) transcripts on D. melanogaster embryos. An estimate of the factor converting immunofluorescence intensity units into molecular numbers is given. The analysis of the eve mRNA's dynamics at the region of the stripe 2 suggests that the promoter site of the gene has two distinct regimes: an earlier phase when it is predominantly activated until a critical time when it becomes mainly repressed. That suggests proposing a stochastic binary model for gene transcription on D. melanogaster embryos. Our model has two random variables: the transcripts number and the state of the source of mRNAs given as active or repressed. We are able to reproduce available experimental data for the average number of transcripts. An analysis of the random fluctuations on the number of eve mRNA's and their consequences on the spatial precision of the stripe 2 is presented. We show that the position of the anterior/posterior borders fluctuate around their average position by ∼1% of the embryo length which is similar to what is found experimentally. The fitting of data by such a simple model suggests that it can be useful to understand the functions of randomness during developmental processes.

In this paper, we propose a thermodynamic mechanism for the formation of transcriptional foci via the joint agglomeration of DNA-looping proteins and protein-binding domains on DNA: The competition between the gain in protein-DNA binding free energy and the entropy loss due to DNA looping is argued to result in an effective attraction between loops. A mean-field approximation can be described analytically via a mapping to a restricted random-graph ensemble having local degree constraints and global constraints on the number of connected components. It shows the emergence of protein clusters containing a finite fraction of all looping proteins. If the entropy loss due to a single DNA loop is high enough, this transition is found to be of first order.

Living cells display a remarkable capacity to compartmentalize their functional biochemistry. A particularly fascinating example is the cell nucleus. Exchange of macromolecules between the nucleus and the surrounding cytoplasm does not involve traversing a lipid bilayer membrane. Instead, large protein channels known as nuclear pores cross the nuclear envelope and regulate the passage of other proteins and RNA molecules. Beyond simply gating diffusion, the system of nuclear pores and associated transport receptors is able to generate substantial concentration gradients, at the energetic expense of guanosine triphosphate (GTP) hydrolysis. In contrast to conventional approaches to demixing such as reverse osmosis or dialysis, the biological system operates continuously, without application of cyclic changes in pressure or solvent exchange. Abstracting the biological paradigm, we examine this transport system as a thermodynamic machine of solution demixing. Building on the construct of free energy transduction and biochemical kinetics, we find conditions for stable operation and optimization of the concentration gradients as a function of dissipation in the form of entropy production.

A general model for the early recognition and colocalization of homologous DNA sequences is proposed. We show, on a thermodynamic ground, how the distance between two homologous DNA sequences is spontaneously regulated by the concentration and affinity of diffusible mediators binding them, which act as a switch between two phases corresponding to independence or colocalization of pairing regions.

Signaling events in eukaryotic cells are often guided by a scaffolding protein. Scaffold proteins assemble multiple proteins in a spatially localized signaling complex and exert numerous physical effects on signaling pathways. To study these effects, we consider a minimal, three-state kinetic model of scaffold mediated kinase activation. We first introduce and apply a path summation technique to obtain approximate solutions to a single molecule master equation that governs protein kinase activation. We then consider exact numerical solutions. We comment on when this approximation is appropriate and then use this analysis to illustrate the competition of processes occurring at many time scales involved in signal transduction in the presence of a scaffold protein. The findings are consistent with recent experiments and simulation data. Our results provide a framework and offer a mechanism for understanding how scaffold proteins can influence the shape of the waiting time distribution of kinase activation and effectively broaden the times over which protein kinases are activated in the course of cell signaling.

Background: Mechanotransduction in bone cells plays a pivotal role in osteoblast differentiation and bone remodelling. Mechanotransduction provides the link between modulation of the extracellular matrix and intracellular actions. By controlling the balance between the intracellular and extracellular domains, the mechanotransduction process determines optimal functionality of the skeletal dynamics, and it is one of the possible causes of osteophatological diseases. Results: Mechanotransduction in a single osteoblast under external mechanical perturbations has been modelled in the agent based framework to reproduce the dynamics of the stochastic reaction diffusion process among molecules in the cytoplasm, nuclear and extracellular domains. The amount of molecules and fluctuations of each molecular class has been analysed in terms of recurrences of critical events. A numerical approach has been developed to invert subordination processes and to extract the directing processes from the molecular signals in order to derive the distribution of recurrence of events. Conclusions: We observed large fluctuations enclose information hidden in the noise which is beyond the dynamic variations of molecular baselines. Studying the system under different parametric conditions and stimuli, the results have shown that the waiting time distributions of each molecule are a signature of the system's dynamics. The behaviours of the molecular waiting times change with the changing of parameters presenting the same variation of patterns for similar interacting molecules and identifying specific alterations for key molecules in the mechanotransduction pathway.