Deciphering clock cell network morphology within the biological master clock, suprachiasmatic nucleus: From the perspective of circadian wave dynamics.

The biological master clock, suprachiasmatic nucleus (of rat and mouse), is composed of ~10,000 clock cells which are heterogeneous with respect to their circadian periods. Despite this inhomogeneity, an intact SCN maintains a very good degree of circadian phase (time) coherence which is vital for sustaining various circadian rhythmic activities, and it is supposedly achieved by not just one but a few different cell-to-cell coupling mechanisms, among which action potential (AP)-mediated connectivity is known to be essential. But, due to technical difficulties and limitations in experiments, so far very little information is available about the morphology of the connectivity at a cellular scale. Building upon this limited amount of information, here we exhaustively and systematically explore a large pool (~25,000) of various network morphologies to come up with some plausible network features of SCN networks. All candidates under consideration reflect an experimentally obtained 'indegree distribution' as well as a 'physical range distribution of afferent clock cells.' Then, importantly, with a set of multitude criteria based on the properties of SCN circadian phase waves in extrinsically perturbed as well as in their natural states, we select out appropriate model networks: Some important measures are, 1) level of phase dispersal and direction of wave propagation, 2) phase-resetting ability of the model networks subject to external circadian forcing, and 3) decay rate of perturbation induced "phase-singularities." The successful, realistic networks have several common features: 1) "indegree" and "outdegree" should have a positive correlation; 2) the cells in the SCN ventrolateral region (core) have a much larger total degree than that of the dorsal medial region (shell); 3) The number of intra-core edges is about 7.5 times that of intra-shell edges; and 4) the distance probability density function for the afferent connections fits well to a beta function. We believe that these newly identified network features would be a useful guide for future explorations on the very much unknown AP-mediated clock cell connectome within the SCN.

Role of senescent cells in the motile behavior of active, non-senescent cells in confluent populations.

Characteristics of cell migration in a confluent population depend on the nature of cell-to-cell interactions as well as cell-intrinsic properties such as the directional persistence in crawling. In addition, biological tissues (or cell cultures) almost always carry anisotropies and they too can significantly affect cell motility. In the light of this viewpoint, the emergence of cellular senescences in a confluent population of active cells raises an interesting question. Cellular senescence is a process through which a cell enters a permanent growth-arrest state and generally exhibits a dramatic body expansion. Therefore, randomly emerging senescent cells transform an initially homogeneous cell population to a "binary mixture" of two distinct cell types. Here, using in vitro cultures of MDA-MB-231 cells we investigate how spatially localized cellular senescence affect the motility of active cells within a confluent population. Importantly, we estimate the intercellular surface energy of the interface between non-senescent and senescent MDA-MB-231 cells by combining the analysis on the motile behaviors of non-senescent cells encircling senescent cells and the result of extensive numerical simulations of a cellular Potts model. We find that the adhesion of normal cells to senescent cells is much weaker than that among normal cells and that the 'arclength' traveled by a normal cell along the boundary of a senescent cell, on average, is several times greater than the persistence length of normal cell in a densely packed homogeneous population. The directional persistent time of normal cell during its contact with a senescent cell also increases significantly. We speculate that the phenomenon could be a general feature associated with senescent cells as the enormous expansion of senescent cell's membrane would inevitably decrease the density of cell adhesion molecules.

Neighbor-enhanced diffusivity in dense, cohesive cell populations.

The dispersal or mixing of cells within cellular tissue is a crucial property for diverse biological processes, ranging from morphogenesis, immune action, to tumor metastasis. With the phenomenon of 'contact inhibition of locomotion,' it is puzzling how cells achieve such processes within a densely packed cohesive population. Here we demonstrate that a proper degree of cell-cell adhesiveness can, intriguingly, enhance the super-diffusive nature of individual cells. We systematically characterize the migration trajectories of crawling MDA-MB-231 cell lines, while they are in several different clustering modes, including freely crawling singles, cohesive doublets of two cells, quadruplets, and confluent population on two-dimensional substrate. Following data analysis and computer simulation of a simple cellular Potts model, which faithfully recapitulated all key experimental observations such as enhanced diffusivity as well as periodic rotation of cell-doublets and cell-quadruplets with mixing events, we found that proper combination of active self-propelling force and cell-cell adhesion is sufficient for generating the observed phenomena. Additionally, we found that tuning parameters for these two factors covers a variety of different collective dynamic states.

Smoking Cessation Pharmacotherapy Utilization and Costs to a Medicaid Managed Care Plan.

BACKGROUND: Medicaid coverage for smoking cessation medications has expanded; however, little research has been conducted to evaluate patient-level changes in medication use over time and its associated economic impact on health plans. OBJECTIVE: The aim of this study was to characterize trends in smoking cessation medication utilization between 2006 and 2017 within a Medicaid population and estimate per-member per-month (PMPM) costs to the health plan. METHODS: This study was a retrospective longitudinal analysis conducted among adult members of a Medicaid managed care plan in California. Pharmacy claims data from January 1, 2006 to December 31, 2017 were analyzed to estimate utilization and cost of smoking cessation medications. Additionally, data from 3164 members who filled prescription(s) for cessation medication(s) in 2017 were evaluated to quantify quit attempts and use of combination therapy. For members who had been prescribed bupropion SR, varenicline, or the nicotine patch, the extent to which the durations of therapy were consistent with the manufacturers' recommended minimum duration of therapy were also assessed. RESULTS: The average PMPM expenditures for smoking cessation medications were approximately US$0.15 in 2017, compared with US$0.01-US$0.03 between 2006 and 2013. In 2017, a total of 3164 members initiated an estimated 3850 quit attempts, most commonly using the nicotine patch (57.5%) or varenicline (32.8%). Combination therapy accounted for 2.9% of quit attempts. The median therapy duration for the nicotine patch, varenicline, and bupropion SR was 28, 30, and 33 days, respectively, and for each of these medications, fewer than half of members filled prescriptions for the minimum recommended duration of therapy. CONCLUSIONS: Pharmacy claims data suggest that despite comprehensive coverage, most beneficiaries are underutilizing smoking cessation agents and are not completing the recommended treatment durations.

Correction: Correction: Two distinct actin waves correlated with turns-and-runs of crawling microglia.

[This corrects the article DOI: 10.1371/journal.pone.0222692.].

Correction: Two distinct actin waves correlated with turns-and-runs of crawling microglia.

[This corrects the article DOI: 10.1371/journal.pone.0220810.].

Two distinct actin waves correlated with turns-and-runs of crawling microglia.

Freely crawling cells are often viewed as randomly moving Brownian particles but they generally exhibit some directional persistence. This property is often related to their zigzag motile behaviors that can be described as a noisy but temporally structured sequence of "runs" and "turns." However, its underlying biophysical mechanism is largely unexplored. Here, we carefully investigate the collective actin wave dynamics associated with the zigzag-crawling movements of microglia (as primary brain immune cells) employing a number of different quantitative imaging modalities including synthetic aperture microscopy and optical diffraction tomography, as well as conventional fluorescence imaging and scanning electron microscopy. Interestingly, we find that microglia exhibit two distinct types of actin waves working at two quite different time scales and locations, and they seem to serve different purposes. One type of actin waves is fast "peripheral ruffles" arising spontaneously with an oscillating period of about 6 seconds at some portion of the leading edge of crawling microglia, where the vigorously biased peripheral ruffles seem to set the direction of a new turn (in 2-D free space). When the cell turning events are inhibited with a physical confinement (in 1-D track), the peripheral ruffles still exist at the leading edge with no bias but showing phase coherence in the cell crawling direction. The other type is "dorsal actin waves" which also exhibits an oscillatory behavior but with a much longer period of around 2 minutes compared to the fast "peripheral ruffles". Dorsal actin waves (whether the cell turning events are inhibited or not) initiate in the lamellipodium just behind the leading edge, travelling down toward the core region of the cell and disappear. Such dorsal wave propagations seem to be correlated with migration of the cell. Thus, we may view the dorsal actin waves are connected with the "run" stage of cell body, whereas the fast ruffles at the leading front are involved in the "turn" stage.

Diversity in the structure of action potential-mediated neural connectivity within rat supra chiasmatic nucleus.

Action potential (AP)-mediated cell-to-cell communication is essential for the frequency-locking and phase-synchronization of the clock cells within the biological master clock, suprachiasmatic nucleus (SCN). Nevertheless, the morphology of its network connectivity is largely unexplored. Here, with an optimized optogenetic light-stimulation and scanning protocol, we report some key characteristics of the inhibitory receptive field (IRF), the area which brings inhibitory synaptic currents to a given target cell, and basic statistics of the inhibitory network connections of rat SCN clock cells. ChR2 transfected, slice cultures of rat SCN were stimulated by a blue power LED light in a repetitive box-scanning modes, while a target cell was whole-cell patched. The registered inhibitory postsynaptic currents, which were brought by light-induced APs of presynaptic neurons, were mostly GABAergic. The sizes and shapes of IRFs of SCN cells were very diverse, and the number of presynaptic cells making up the IRF of a given target cell followed an exponential distribution with an average value of 8.9 approximately, according to our clustering analysis which is based on a hybrid measure D, combining the physical distance r and the difference in the current amplitudes of two different sites. Although this estimate inevitably depends on the construct of the measure D, it is found not so sensitive on the parameter w, which weighs the relative significance of the current amplitude different with respect to the physical distance r: The average number of presynaptic neurons varies < 26% over a significant range of 0 < w < 30. On average, the presynaptic connection number density around a target cell falls off as an exponentially decreasing function of r. But, its space constant (~210.7 µm) is quite large that long-range (>210.7 µm) neural connections are abundant (>66.9%) within the SCN.

Encoding information into autonomously bursting neural network with pairs of time-delayed pulses.

Biological neural networks with many plastic synaptic connections can store external input information in the map of synaptic weights as a form of unsupervised learning. However, the same neural network often produces dramatic reverberating events in which many neurons fire almost simultaneously - a phenomenon coined as 'population burst.' The autonomous bursting activity is a consequence of the delicate balance between recurrent excitation and self-inhibition; as such, any periodic sequences of burst-generating stimuli delivered even at a low frequency (~1 Hz) can easily suppress the entire network connectivity. Here we demonstrate that 'Δt paired-pulse stimulation', can be a novel way for encoding spatially-distributed high-frequency (~10 Hz) information into such a system without causing a complete suppression. The encoded memory can be probed simply by delivering multiple probing pulses and then estimating the precision of the arrival times of the subsequent evoked recurrent bursts.

Senescent tumor cells building three-dimensional tumor clusters.

Cellular senescence, a permanent cell-cycle arrest, is a common yet intriguing phenomenon, in which its beneficial significance for biological organisms has only begun to be explored. Among others, senescent cells are able to transform tissue structures around them. Tumor cells, whose hallmark is their ability to proliferate indefinitely, are not free from the phenomenon. Here, we report a remarkable observation where senescent cells in a dense mono-layer of breast cancer colony act as aggregating centers for non-senescent cells in their vicinity. Consequently, the senescent cells actively form localized 3D cell-clusters in a confluent 2D tumor layer. The biophysical mechanism underpinning the surprising phenomenon primarily involves mitotic cell-rounding, dynamic and differential cell attachments, and cellular chemotaxis. By incorporating these few biophysical factors, we were able to recapitulate the experimental observation via a cellular Potts Model.

Single-shot digital holographic microscopy for quantifying a spatially-resolved Jones matrix of biological specimens.

Field-based polarization measurements are essential for the completeness of information when exploiting the complex nature of optical responses of target objects. Here, we demonstrate digital holographic microscopy for quantifying a polarization-sensitive map of an object with a single-shot measurement. Using the image-splitting device generating four different copies of an object image and a separate reference beam of an off-axis configuration enables single-shot and multi-imaging capability. With the use of two polarization filters, four complex field images containing an object's polarization response are obtained simultaneously. With this method, we can construct a complete set of 2-by-2 Jones matrix at every single point of the object's images, and thus clearly visualize the anisotropic structures of biological tissues with low level of birefringence. This method will facilitate the high-precision measurements for fast dynamics of the polarization properties of biological specimens.

Single-shot and phase-shifting digital holographic microscopy using a 2-D grating.

We demonstrate digital holographic microscopy that, while being based on phase-shifting interferometry, is capable of single-shot measurements. A two-dimensional (2-D) diffraction grating placed in a Fourier plane of a standard in-line holographic phase microscope generates multiple copies of a sample image on a camera sensor. The identical image copies are spatially separated with different overall phase shifts according to the diffraction orders. The overall phase shifts are adjusted by controlling the lateral position of the grating. These phase shifts are then set to be multiples of π/2. Interferograms composed of four image copies combined with a parallel reference beam are acquired in a single shot. The interferograms are processed through a phase-shifting algorithm to produce a single complex image. By taking advantage of the higher sampling capacity of the in-line holography, we can increase the imaging information density by a factor of 3 without compromising the imaging acquisition speed.

Enhancement of Chemotactic Cell Aggregation by Haptotactic Cell-To-Cell Interaction.

The crawling of biological cell is a complex phenomenon involving various biochemical and mechanical processes. Some of these processes are intrinsic to individual cells, while others pertain to cell-to-cell interactions and to their responses to extrinsically imposed cues. Here, we report an interesting aggregation dynamics of mathematical model cells, when they perform chemotaxis in response to an externally imposed global chemical gradient while they influence each other through a haptotaxis-mediated social interaction, which confers intriguing trail patterns. In the absence of the cell-to-cell interaction, the equilibrium population density profile fits well to that of a simple Keller-Segal population dynamic model, in which a chemotactic current density [Formula: see text] competes with a normal diffusive current density [Formula: see text], where p and ρ refer to the concentration of chemoattractant and population density, respectively. We find that the cell-to-cell interaction confers a far more compact aggregation resulting in a much higher peak equilibrium cell density. The mathematical model system is applicable to many biological systems such as swarming microglia and neutrophils or accumulating ants towards a localized food source.

Multi-stability of circadian phase wave within early postnatal suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is a group of cells that functions as a biological master clock. In different SCN cells, oscillations of biochemical markers such as the expression-level of clock genes, are not synchronized but instead form slow circadian phase waves propagating over the whole cell population spatio-temporal structure is a fixed property set by the anatomy of a given SCN. Here, we show that this is not the case in early postnatal SCN. Earlier studies presumed that their Based on bioluminescence imaging experiments with Per2-Luciferase mice SCN cultures which guided computer simulations of a realistic model of the SCN, we demonstrate that the wave is not unique but can be in various modes including phase- coherent oscillation, crescent-shaped wave, and most notably, a rotating pinwheel wave that conceptually resembles a wall clock with a rotating hand. Furthermore, mode transitions can be induced by a pulse of 38.5 °C temperature perturbation. Importantly, the waves support a significantly different period, suggesting that neither a spatially-fixed phase ordering nor a specialized pacemaker having a fixed period exist in these studied SCNs. These results lead to new important questions of what the observed multi-stability means for the proper function of an SCN and its arrhythmia.

Coherence resonance in bursting neural networks.

Synchronized neural bursts are one of the most noticeable dynamic features of neural networks, being essential for various phenomena in neuroscience, yet their complex dynamics are not well understood. With extrinsic electrical and optical manipulations on cultured neural networks, we demonstrate that the regularity (or randomness) of burst sequences is in many cases determined by a (few) low-dimensional attractor(s) working under strong neural noise. Moreover, there is an optimal level of noise strength at which the regularity of the interburst interval sequence becomes maximal-a phenomenon of coherence resonance. The experimental observations are successfully reproduced through computer simulations on a well-established neural network model, suggesting that the same phenomena may occur in many in vivo as well as in vitro neural networks.

Serotonin-2C receptor involved serotonin-induced Ca²âº mobilisations in neuronal progenitors and neurons in rat suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, undergoes serotonergic regulation, but the underlying mechanisms remain obscure. Here, we generated a subclone of an SCN progenitor cell line expressing Ca(2+) sensors (SCN2.2YC) and compared its 5-HT receptor signalling with that of rat SCN neurons in brain slices. SCN2.2YC cells expressed 5-HT1A/2A/2B/2C, but not 5A/7, while all six subtypes were expressed in SCN tissues. High K(+) or 5-HT increased cytosolic Ca(2+) in SCN2.2YC cells. The 5-HT responses were inhibited by ritanserin and SB-221284, but resistant to WAY-100635 and RS-127445, suggesting predominant involvement of 5-HT2C for Ca(2+) mobilisations. Consistently, Ca(2+) imaging and voltage-clamp electrophysiology using rat SCN slices demonstrated post-synaptic 5-HT2C expression. Because 5-HT2C expression was postnatally increased in the SCN and 5-HT-induced Ca(2+) mobilisations were amplified in differentiated SCN2.2YC cells and developed SCN neurons, we suggest that this signalling development occurs in accordance with central clock maturations.

Modulating the precision of recurrent bursts in cultured neural networks.

Synchronized bursts are a very common feature in biological neural networks, and they play an important role in various brain functions and neurological diseases. This Letter investigates "recurrent synchronized bursts" induced by a single pulse stimulation in cultured networks of rat cortical neurons. We look at how the precision in their arrival times can be modified by a noble time-delayed stimulation protocol, which we term as "Δt training." The emergence of recurrent bursts and the change of the precision in their arrival times can be explained by the stochastic resonance of a damped, subthreshold, neural oscillation.

Circadian waves of cytosolic calcium concentration and long-range network connections in rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the master clock in mammals governing the daily physiological and behavioral rhythms. It is composed of thousands of clock cells with their own intrinsic periods varying over a wide range (20-28 h). Despite this heterogeneity, an intact SCN maintains a coherent 24 h periodic rhythm through some cell-to-cell coupling mechanisms. This study examined how the clock cells are connected to each other and how their phases are organized in space by monitoring the cytosolic free calcium ion concentration ([Ca(2+)](c)) of clock cells using the calcium-binding fluorescent protein, cameleon. Extensive analysis of 18 different organotypic slice cultures of the SCN showed that the SCN calcium dynamics is coordinated by phase-synchronizing networks of long-range neurites as well as by diffusively propagating phase waves. The networks appear quite extensive and far-reaching, and the clock cells connected by them exhibit heterogeneous responses in their amplitudes and periods of oscillation to tetrodotoxin treatments. Taken together, our study suggests that the network of long-range cellular connectivity has an important role for the SCN in achieving its phase and period coherence.

In situ analysis of cisplatin binding to DNA: the effects of physiological ionic conditions.

Platinum-based anti-cancer drugs form a major family of cancer chemotherapeutic agents. Cisplatin, the first member of the family, remains a potent anti-cancer drug and exhibits its clinical effect by inducing local DNA kinks and subsequently interfering with DNA metabolism. Although its mechanism is reasonably well understood, effects of intracellular ions on cisplatin activity are left to be elucidated because cisplatin binding to DNA, thus its drug efficacy, is modified by various ions. One such issue is the effect of carbonate ions: cisplatin binding to DNA is suppressed under physiological carbonate conditions. Here, we examined the role of common cellular ions (carbonate and chloride) by measuring cisplatin binding in relevant physiological buffers via a DNA micromanipulation technique. Using two orthogonal single-molecule methods, we succeeded in detecting hidden monofunctional adducts (kink-free, presumably clinically inactive form) and clearly showed that the major effect of carbonates was to form such adducts and to prevent them from converting to bifunctional adducts (kinked, clinically active). The chloride-rich environment also led to the formation of monofunctional adducts. Our approach is widely applicable to the study of the transient behaviours of various drugs and proteins that bind to DNA in different modes depending on various physical and chemical factors such as tension, torsion, ligands, and ions.