Tomasz S. Kaminski

Bacterial Growth and Adaptation in Microdroplet Chemostats

We describe herein microfluidic technology for manipulating and monitoring continuous growth of populations of bacteria. A system consisting of approximately ten input and output channels controls more than 100 microdroplet chemostats and enables the manipulation of chemical factors in each microchemostat independently over time. Herein, we characterize the dynamics of bacterial populations in microdroplet chemostats and cellular responses to a range of stable or changing antibiotic concentrations. This method allows for parallel, long-term studies of microbial ecology, physiology, evolution, and adaptation to chemical environments. The introduction of the chemostat by Leo Szilard was a milestone in the field of microbiology. Chemostats facilitate the continuous culture of bacteria, yeast, and algae by continuously replenishing a constant volume of fluid to maintain specific concentrations of cells and growth factors. Chemostats have facilitated a wide-range of studies, including microbial ecology, predator–prey dynamics, and the evolution of drug resistance. The consumption of large quantities of reagents and the significant operational challenges of traditional chemostats limit their use. Single-phase, microfluidic versions of chemostats minimize incubation volumes, and yet are limited by their complexity: the proportionality between the number of input/ output controls and the number of chemostats hamper large scale parallelization. Single-phase microfluidic systems are prone to biofilm formation, which makes them either singleuse devices or requiring additional steps to minimize cell adhesion. Droplet microfluidics offer a unique solution to creating many parallel chemostats. The earliest example of this technology in microbiology was first demonstrated by Joshua Lederberg nearly 60 years ago. In the interim, the field of microfluidics solved many of the technical challenges associated with using this approach to study microbes. Compartmentalizing cells and nutrients in microdroplets of liquid can reduce the complexity and cost of operating many parallel chemostats. Recently, bacteria have been incubated in droplets in channels over short time intervals, however sustained cell growth over hundreds of generations in a series of fully addressable microdroplets has not been possible. Herein, we describe an automated microdroplet system that transcends existing challenges and enables users to manipulate the chemical composition of droplets for longterm bacterial studies. The microfluidic system (Figure 1) performs three functions: 1) formation of microdroplets containing cells, reagents, and soluble growth factors; 2) cycling microdroplets for cell incubation and monitoring; and 3) splitting and fusing microdroplets to control the concentration of chemical factors over time. After loading the reservoirs with liquid samples, we used a source of pressure and external valves to regulate the flow of

Formation and structure of PEI/DNA complexes: quantitative analysis

Controlled formation of gene delivery complexes (DNA and a vector, usually a cationic polymer) is one of the key challenges in developing efficient gene delivery systems. The researchers focused their procedures on the ratio of vector to DNA, neglecting the influence of concentration on the complex formation process. In this study we show, by studying the association of polyethylenimine (PEI) and 66-base pair (bp) DNA fragments, that the concentration of the gene delivery system greatly influences the formation of PEI/DNA complexes even at a fixed PEI/DNA ratio. We find that the charge and the size of PEI/DNA complexes are increasing functions of their concentration even in a highly dilute regime of concentrations. The number of PEI/DNA molecules in a complex was calculated from the measured charge and electrophoretic mobility. We established a model, on the basis of Smoluchowski theory, to explain the relation between the concentration and the size of PEI/DNA complexes. We analyzed the structure of the complexes and found out that a large proportion of space in the PEI/DNA complexes is occupied by the solvent. This study indicates that the influence of concentration should be seriously considered in gene delivery studies, since large PEI/DNA complexes can be prepared by scaling up their concentration simultaneously without increasing the dosage of PEI.

Characterization of Caulobacter crescentus FtsZ Protein Using Dynamic Light Scattering

Background: Self-assembly of the tubulin-homologue FtsZ is critical in bacterial cell division. Results: Dynamic light scattering (DLS) measurements provide insight into the kinetics and stable length of Caulobacter crescentus FtsZ in vitro. Conclusion: C. crescentus FtsZ forms short linear polymers in solution with the assembly rate depending on the concentrations of GTP and GDP. Significance: DLS is a valuable technique for studying the polymerization of cytoskeletal proteins. The self-assembly of the tubulin homologue FtsZ at the mid-cell is a critical step in bacterial cell division. We introduce dynamic light scattering (DLS) spectroscopy as a new method to study the polymerization kinetics of FtsZ in solution. Analysis of the DLS data indicates that the FtsZ polymers are remarkably monodisperse in length, independent of the concentrations of GTP, GDP, and FtsZ monomers. Measurements of the diffusion coefficient of the polymers demonstrate that their length is remarkably stable until the free GTP is consumed. We estimated the mean size of the FtsZ polymers within this interval of stable length to be between 9 and 18 monomers. The rates of FtsZ polymerization and depolymerization are likely influenced by the concentration of GDP, as the repeated addition of GTP to FtsZ increased the rate of polymerization and slowed down depolymerization. Increasing the FtsZ concentration did not change the size of FtsZ polymers; however, it increased the rate of the depolymerization reaction by depleting free GTP. Using transmission electron microscopy we observed that FtsZ forms linear polymers in solutions which rapidly convert to large bundles upon contact with surfaces at time scales as short as several seconds. Finally, the best studied small molecule that binds to FtsZ, PC190723, had no stabilizing effect on Caulobacter crescentus FtsZ filaments in vitro, which complements previous studies with Escherichia coli FtsZ and confirms that this class of small molecules binds Gram-negative FtsZ weakly.

Influence of nano-viscosity and depletion interactions on cleavage of DNA by enzymes in glycerol and poly(ethylene glycol) solutions: qualitative analysis

Biochemical reactions in living systems take place in an environment crowded by various macromolecules and ligands. Therefore experimental data obtained in buffer do not reflect in vivo conditions. We have used glycerol, poly(ethylene glycol) (PEG) 6000 and PEG 8 M solutions to investigate the influence of the crowded environment on cleavage of plasmid DNA by restriction enzyme HindIII. PEG 6000 solution can effectively slow down the cleavage process. However, neither PEG 8 M solution of the same viscosity as PEG 6000 solution nor glycerol solution of the same concentration as PEG 6000 solution slows the cleavage of DNA appreciably. The viscosity experienced by the biomolecules (here called nano-viscosity) and aggregation induced by the depletion interactions between DNA molecules in polymer solution (PEG 6000) are two factors responsible for slow cleavage of DNA. We have ruled out the change of pH and denaturation of HindIII as possible sources for the effect.

FOXO1 is a TXN- and p300-dependent sensor and effector of oxidative stress in diffuse large B-cell lymphomas characterized by increased oxidative metabolism

Molecular profiling has led to identification of subtypes of diffuse large B-cell lymphomas (DLBCLs) differing in terms of oncogenic signaling and metabolic programs. The OxPhos-DLBCL subtype is characterized by enhanced mitochondrial oxidative phosphorylation. As increased oxidative metabolism leads to overproduction of potentially toxic reactive oxygen species (ROS), we sought to identify mechanisms responsible for adaptation of OxPhos cells to these conditions. Herein, we describe a mechanism involving the FOXO1–TXN–p300 redox-dependent circuit protecting OxPhos-DLBCL cells from ROS toxicity. We identify a BCL6-dependent transcriptional mechanism leading to relative TXN overexpression in OxPhos cells. We found that OxPhos cells lacking TXN were uniformly more sensitive to ROS and doxorubicin than control cells. Consistent with this, the overall survival of patients with high TXN mRNA expression, treated with doxorubicin-containing regimens, is significantly shorter than of those with low TXN mRNA expression. TXN overexpression curtails p300-mediated FOXO1 acetylation and its nuclear translocation in response to oxidative stress, thus attenuating FOXO1 transcriptional activity toward genes involved in apoptosis and cell cycle inhibition. We also demonstrate that FOXO1 knockdown in cells with silenced TXN expression markedly reduces ROS-induced apoptosis, indicating that FOXO1 is the major sensor and effector of oxidative stress in OxPhos-DLBCLs. These data highlight dynamic, context-dependent modulation of FOXO1 tumor-suppressor functions via acetylation and reveal potentially targetable vulnerabilities in these DLBCLs.

Rational design of digital assays.

Optimum algorithm for digital assays treats chemical compartments as bits of probabilistic information and arranges these bits in a fractional positional system. Maximization of information gain reduces, by orders of magnitude, the number of partitions required to achieve the requested dynamic range and precision of the assay. The method simplifies the execution of digital analytical methods providing for more accessible use of absolute quantization in research and in diagnostics.

Dodecylresorufin (C12R) Outperforms Resorufin in Microdroplet Bacterial Assays

This paper proves that dodecylresorufin (C12R) outperforms resorufin (the conventional form of this dye) in droplet microfluidic bacterial assays. Resorufin is a marker dye that is widely used in different fields of microbiology and has increasingly been applied in droplet microfluidic assays and experiments. The main concern associated with resorufin in droplet-based systems is dye leakage into the oil phase and neighboring droplets. The leakage decreases the performance of assays because it causes averaging of the signal between the positive (bacteria-containing) and negative (empty) droplets. Here we show that C12R is a promising alternative to conventional resorufin because it maintains higher sensitivity, specificity, and signal-to-noise ratio over time. These characteristics make C12R a suitable reagent for droplet digital assays and for monitoring of microbial growth in droplets.

An Automated Microfluidic System for the Generation of Droplet Interface Bilayer Networks

Networks of droplets, in which aqueous compartments are separated by lipid bilayers, have shown great potential as a model for biological transmembrane communication. We present a microfluidic system which allows for on-demand generation of droplets that are hydrodynamically locked in a trapping structure. As a result, the system enables the formation of a network of four droplets connected via lipid bilayers and the positions of each droplet in the network can be controlled thanks to automation of microfluidic operations. We perform electrophysiological measurements of ionic currents indicating interactions between nanopores and small molecules to prove the potential of the device in screening of the inhibitors acting on membrane proteins. We also demonstrate, for the first time, a microfluidic droplet interface bilayer (DIB) system in which the testing of inhibitors can be performed without direct contact between the tested sample and the electrodes recording picoampere currents.

Human and the beast—Flight and aggressive responses of European bison to human disturbance

Large mammals are often a source of conflict, and consequently there has been increasing interest in close encounters with them. Knowledge of wildlife responses to human disturbance is crucial for the management of increasing and expanding populations of large animals. We investigated flight initiation distance (FID) and aggressive responses of the European bison–the largest terrestrial mammal of Europe–to human disturbance in the Białowieża Forest (NE Poland). When encountered by humans, bison usually flee. Aggression was observed in only 0.4% of approach attempts. Mean FID was 77±46 m and was influenced by habitat, sex, and supplementary feeding intensity. Females showed greater timidity than males, FID was lower in forest than in open habitats, and supplementary feeding caused a drop in FID. In 84.5% of all documented aggression cases, bison attacks were provoked by humans approaching too close to the bison or by deliberate scaring them. Males were more aggressive than females, and attacked mainly during the rut, while females attacked during the winter and calving. Bison attacked in built-up areas significantly more often than expected. The mean critical distance of attacks was 21±2 m. Most attacks took the form of a short chase preceded by warning signs. Goring was observed in 22.7% of all aggression cases and no fatalities were recorded. Our study shows that bison are not dangerous animals and only manifest aggression in response to prolonged disturbance at close ranges. The education of people and recommendations for minimum approach distances should ensure a low rate of disturbance and safety when encountering large mammals.

Automated Droplet Microfluidic Chips for Biochemical Assays

After 20 years of research on microfluidic systems a vast expertise is available on automation of single phase flows through the use of mechanical actuation [1, 2] or through the use of electrokinetic effects [3, 4]. These systems are perfectly suited for a range of applications but are inherently inefficient in handling massively large numbers of processes due to correspondingly large number of input/output controls that at best scales logarithmically in the number of processes.