Plasmid copy number noise in monoclonal populations of bacteria.

Plasmids are extra chromosomal DNA that can confer to their hosts' supplementary characteristics such as antibiotic resistance. Plasmids code for their copy number through their own replication frequency. Even though the biochemical networks underlying the plasmid copy number (PCN) regulation processes have been studied and modeled, no measurement of the heterogeneity in PCN within a whole population has been done. We have developed a fluorescent-based measurement system, which enables determination of the mean and noise in PCN within a monoclonal population of bacteria. Two different fluorescent protein reporters were inserted: one on the chromosome and the other on the plasmid. The fluorescence of these bacteria was measured with a microfluidic flow cytometry device. We show that our measurements are consistent with known plasmid characteristics. We find that the partitioning system lowers the PCN mean and standard deviation. Finally, bacterial populations were allowed to grow without selective pressure. In this case, we were able to determine the plasmid loss rate and growth inhibition effect.

Automated imaging of neuronal activity in freely behaving Caenorhabditis elegans.

In order to understand how neuronal circuits control locomotory patterns it is necessary to record neuronal activity of freely behaving animals. Here, using a new automated system for simultaneous recording of behavior and neuronal activity in freely moving Caenorhabditis elegans on standard agar plates, we show that spontaneous reversals from forward to backward locomotion reflect precisely the activity of the AVA command interneurons. We also witness spontaneous activity transients in the PLM sensory neurons during free behavior of the worm in standard conditions. We show that these activity transients are coupled to short spontaneous forward accelerations of the worm.

Inference of plasmid-copy-number mean and noise from single-cell gene expression data.

Plasmids are extrachromosomal DNA molecules which code for their own replication. We previously reported a setup using genes coding for fluorescent proteins of two colors that allowed us, using a simple model, to extract the plasmid-copy-number noise in a monoclonal population of bacteria [J. Wong Ng, Phys. Rev. E 81, 011909 (2010)]. Here we present a detailed calculation relating this noise to the measured levels of fluorescence, taking into account all sources of fluorescence fluctuations: not only the fluctuation of gene expression as in the simple model but also the growth and division of bacteria, the nonuniform distribution of their ages, the random partition of proteins at divisions, and the replication and partition of plasmids and chromosome. We show how to use the chromosome as a reference, which helps extracting the plasmid-copy-number noise in a self-consistent manner.

Molecular and sensory basis of a food related two-state behavior in C. elegans.

Most animals display multiple behavioral states and control the time allocation to each of their activity phases depending on their environment. Here we develop a new quantitative method to analyze Caenorhabditis elegans behavioral states. We show that the dwelling and roaming two-state behavior of C. elegans is tightly controlled by the concentration of food in the environment of the animal. Sensory perception through the amphid neurons is necessary to extend roaming phases while internal metabolic perception of food nutritional value is needed to induce dwelling. Our analysis also shows that the proportion of time spent in each state is modulated by past nutritional experiences of the animal. This two-state behavior is regulated through serotonin as well as insulin and TGF-beta signaling pathways. We propose a model where food nutritional value is assessed through internal metabolic signaling. Biogenic amines signaling could allow the worm to adapt to fast changes in the environment when peptide transcriptional pathways may mediate slower adaptive changes.

Propagation of fluorescent viruses in growing plaques.

To study virus propagation, we have developed a method by which the propagation of the Lambda bacteriophage can be observed and quantified. This is done by creating a fusion protein of the capsid protein gpD and the enhanced yellow fluorescent protein (EYFP). We show that this fusion allows capsid formation and that the modified viruses propagate on a surface covered with host bacteria thus forming fluorescent plaques. The intensity of fluorescence in a growing plaque determines the distribution of phages. This provides a new tool to study the propagation of infection at the microscopic level.

Dynamics of excitatory synaptic components in sustained firing at low rates.

Sustained firing is necessary for the persistent activity associated with working memory. The relative contributions of the reverberation of excitation and of the temporal dynamics of the excitatory postsynaptic potential (EPSP) to the maintenance of activity are difficult to evaluate in classical preparations. We used simplified models of synchronous excitatory networks, hippocampal autapses and pairs, to study the synaptic mechanisms underlying firing at low rates. Calcium imaging and cell attached recordings showed that these neurons spontaneously fired bursts of action potentials that lasted for seconds over a wide range of frequencies. In 2-wk-old cells, the median firing frequency was low (11 +/- 8.8 Hz), whereas in 3- to 4-wk-old cells, it decreased to a very low value (2 +/- 1.3 Hz). In both cases, we have shown that the slowest synaptic component supported firing. In 2-wk-old autapses, antagonists of N-methyl-d-aspartate receptors (NMDARs) induced rare isolated spikes showing that the NMDA component of the EPSP was essential for bursts at low frequency. In 3- to 4-wk-old neurons, the very low frequency firing was maintained without the NMDAR activation. However EGTA-AM or alpha-methyl-4-carboxyphenylglycine (MCPG) removed the very slow depolarizing component of the EPSP and prevented the sustained firing at very low rate. A metabotropic glutamate receptor (mGluR)-activated calcium sensitive conductance is therefore responsible for a very slow synaptic component associated with firing at very low rate. In addition, our observations suggested that the asynchronous release of glutamate might participate also in the recurring bursting.

Overstretching and force-driven strand separation of double-helix DNA.

We analyze whether the "overstretched," or "S" form of double-stranded DNA consists of essentially separated, or essentially interacting, polynucleotide strands. Comparison of force-extension data for S-DNA and single-stranded DNA shows S-DNA to be distinct from both double helix and single-stranded forms. We use a simple thermodynamical model for tension-melted double-stranded DNA, which indicates that the overstretching transition near 65 piconewtons cannot be explained in terms of conversion of double helix to noninteracting polynucleotide strands. However, the single-strand-like response observed in some experiments can be explained in terms of "unpeeling" of large regions of one strand, starting from nicks on the original double helix. We show that S-DNA becomes unstable to unpeeling at large forces, and that at low ionic strength, or for weakly base-paired sequences, unpeeling can preempt formation of S-DNA. We also analyze the kinetics of unpeeling including the effect of sequence-generated free energy inhomogeneity. We find that strongly base-paired regions generate large barriers that stabilize DNA against unpeeling. For long genomic sequences, these barriers to unpeeling cannot be kinetically crossed until force exceeds approximately 150 piconewtons.

Constrained synaptic connectivity in functional mammalian neuronal networks grown on patterned surfaces.

The use of ordered neuronal networks in vitro is a promising approach to study the development and the activity of small neuronal assemblies. However, in previous attempts, sufficient growth control and physiological maturation of neurons could not be achieved. Here we describe an original protocol in which polylysine patterns confine the adhesion of cellular bodies to prescribed spots and the neuritic growth to thin lines. Hippocampal neurons in these networks are maintained healthy in serum free medium up to 5 weeks in vitro. Electrophysiology and immunochemistry show that neurons exhibit mature excitatory and inhibitory synapses and calcium imaging reveals spontaneous activity of neurons in isolated networks. We demonstrate that neurons in these geometrical networks form functional synapses preferentially to their first neighbors. We have, therefore, established a simple and robust protocol to constrain both the location of neuronal cell bodies and their pattern of connectivity. Moreover, the long term maintenance of the geometry and the physiology of the networks raises the possibility of new applications for systematic screening of pharmacological agents and for electronic to neuron devices.