Particle image velocimetry and infrared thermography in a levitated droplet with nanosilica suspensions

Preferential accumulation and agglomeration kinetics of nanoparticles suspended in an acoustically levitated water droplet under radiative heating has been studied. Particle image velocimetry performed to map the internal flow field shows a single cell recirculation with increasing strength for decreasing viscosities. Infrared thermography and high speed imaging show details of the heating process for various concentrations of nanosilica droplets. Initial stage of heating is marked by fast vaporization of liquid and sharp temperature rise. Following this stage, aggregation of nanoparticles is seen resulting in various structure formations. At low concentrations, a bowl structure of the droplet is dominant, maintained at a constant temperature. At high concentrations, viscosity of the solution increases, leading to rotation about the levitator axis due to the dominance of centrifugal motion. Such complex fluid motion inside the droplet due to acoustic streaming eventually results in the formation of a ring structure. This horizontal ring eventually reorients itself due to an imbalance of acoustic forces on the ring, exposing larger area for laser absorption and subsequent sharp temperature rise.

Toll-Like Receptors 7, 8, and 9 Expression and Function in Primary Human Cervical Cancer Langerhans Cells Evidence of Anergy

Objective: Human papillomavirus oncoproteins E6 and E7 down modulate Toll-like receptor (TLR) 9 expression in infected keratinocytes. We explored the status of expression and function of TLR7, TLR8, and TLR9 in primary human Langerhans cells (LCs) isolated from cervical tumors. Methodology: Single-cell suspensions were made from fresh tissues of squamous cell carcinoma (International Federation of Gynecology and Obstetrics stage IB2); myeloid dendritic cells were purified using CD1c magnetic activated cell separation kits. Langerhans cells were further flow sorted into CD1a(+)CD207(+) cells. Acute monocytic leukemia cell line THP-1-derived LCs (moLCs) formed the controls. mRNA from flow-sorted LCs was reverse transcribed to cDNA and TLR7, TLR8, and TLR9 amplified. Monocyte-derived Langerhans cells and cervical tumor LCs were stimulated with TLR7, TLR8, and TLR9 ligands. Culture supernatants were assayed for interleukin (IL) 1 beta, IL-6, IL-10, IL-12p70, interferon (IFN) alpha, interferon gamma, and tumor necrosis factor (TNF) alpha by Luminex multiplex bead array. Human papillomavirus was genotyped. Results: We have for the first time demonstrated that the acute monocytic leukemia cell line THP-1 can be differentiated into LCs in vitro. Although these moLCs. expressed all the 3 TLRs, tumor LCs expressed TLR7 and TLR8, but uniformly lacked TLR9. Also, moLCs secreted IL-6, IL-1 beta, and tumor necrosis factor alpha to TLR8 ligand and interferon alpha in response to TLR9 ligand; in contrast, tumor LCs did not express any cytokine to any of the 3 TLR ligands. Human papillomavirus type 16 was one of the common human papillomavirus types in all cases. Conclusions: Cervical tumor LCs lacked TLR9 expression and were functionally anergic to all the 3: TLR7, TLR8, and TLR9 ligands, which may play a crucial role in immune tolerance. The exact location of block(s) in TLR7 and TLR8 signaling needs to be investigated, which would have important immunotherapeutic implications.

A Study of Early Afterdepolarizations in a Model for Human Ventricular Tissue

Sudden cardiac death is often caused by cardiac arrhythmias. Recently, special attention has been given to a certain arrhythmogenic condition, the long-QT syndrome, which occurs as a result of genetic mutations or drug toxicity. The underlying mechanisms of arrhythmias, caused by the long-QT syndrome, are not fully understood. However, arrhythmias are often connected to special excitations of cardiac cells, called early afterdepolarizations (EADs), which are depolarizations during the repolarizing phase of the action potential. So far, EADs have been studied mainly in isolated cardiac cells. However, the question on how EADs at the single-cell level can result in fibrillation at the tissue level, especially in human cell models, has not been widely studied yet. In this paper, we study wave patterns that result from single-cell EAD dynamics in a mathematical model for human ventricular cardiac tissue. We induce EADs by modeling experimental conditions which have been shown to evoke EADs at a single-cell level: by an increase of L-type Ca currents and a decrease of the delayed rectifier potassium currents. We show that, at the tissue level and depending on these parameters, three types of abnormal wave patterns emerge. We classify them into two types of spiral fibrillation and one type of oscillatory dynamics. Moreover, we find that the emergent wave patterns can be driven by calcium or sodium currents and we find phase waves in the oscillatory excitation regime. From our simulations we predict that arrhythmias caused by EADs can occur during normal wave propagation and do not require tissue heterogeneities. Experimental verification of our results is possible for experiments at the cell-culture level, where EADs can be induced by an increase of the L-type calcium conductance and by the application of I-Kr blockers, and the properties of the emergent patterns can be studied by optical mapping of the voltage and calcium.

Competing views on cancer

Despite intense research efforts that have provided enormous insight, cancer continues to be a poorly understood disease. There has been much debate over whether the cancerous state can be said to originate in a single cell or whether it is a reflection of aberrant behaviour on the part of a `society of cells'. This article presents, in the form of a debate conducted among the authors, three views of how the problem might be addressed. We do not claim that the views exhaust all possibilities. These views are (a) the tissue organization field theory (TUFT) that is based on a breakdown of tissue organization involving many cells from different embryological layers, (b) the cancer stem cell (CSC) hypothesis that focuses on genetic and epigenetic changes that take place within single cells, and (c) the proposition that rewiring of the cell's protein interaction networks mediated by intrinsically disordered proteins (IDPs) drives the tumorigenic process. The views are based on different philosophical approaches. In detail, they differ on some points and agree on others. It is left to the reader to decide whether one approach to understanding cancer appears more promising than the other.

Shape Memory Alloy based Active Chiral Composite Cells

Wing morphing is one of the emerging methodology towards improving aerodynamic efficiency of flight vehicle structures. In this paper a morphing structural element is designed and studied which has its origin in the well known chiral structures. The new aspect of design and functionality explored in this paper is that the chiral cell is actuated using thermal Shape Memory Alloy (SMA) actuator wires to provide directional motion. Such structure utilizes the potential of different actuations concepts based on actuator embedded in the chiral structure skin. This paper describes a new class of chiral cell structure with integrated SMA wire for actuation. Chiral topological constructs are obtained by considering passive and active load path decoupling and sub-optimal shape changes. Single cell of chiral honeycomb with actuators are analyzed using finite element simulation results and experiments. To this end, a multi-cell plan-form is characterized showing interesting possibilities in structural morphing applications. The applicability of the developed chiral cell to flexible wing skin, variable stiffness based design and controlling longitudinal-to-transverse stiffness ratio are discussed.

Performance of OFDM Systems With Best-m Feedback, Scheduling, and Delays for Uniformly Correlated Subchannels

Contemporary cellular standards, such as Long Term Evolution (LTE) and LTE-Advanced, employ orthogonal frequency-division multiplexing (OFDM) and use frequency-domain scheduling and rate adaptation. In conjunction with feedback reduction schemes, high downlink spectral efficiencies are achieved while limiting the uplink feedback overhead. One such important scheme that has been adopted by these standards is best-m feedback, in which every user feeds back its m largest subchannel (SC) power gains and their corresponding indices. We analyze the single cell average throughput of an OFDM system with uniformly correlated SC gains that employs best-m feedback and discrete rate adaptation. Our model incorporates three schedulers that cover a wide range of the throughput versus fairness tradeoff and feedback delay. We show that, for small m, correlation significantly reduces average throughput with best-m feedback. This result is pertinent as even in typical dispersive channels, correlation is high. We observe that the schedulers exhibit varied sensitivities to correlation and feedback delay. The analysis also leads to insightful expressions for the average throughput in the asymptotic regime of a large number of users.

Turbulent states and their transitions in mathematical models for ventricular tissue: The effects of random interstitial fibroblasts

We study the dynamical behaviors of two types of spiral-and scroll-wave turbulence states, respectively, in two-dimensional (2D) and three-dimensional (3D) mathematical models, of human, ventricular, myocyte cells that are attached to randomly distributed interstitial fibroblasts; these turbulence states are promoted by (a) the steep slope of the action-potential-duration-restitution (APDR) plot or (b) early afterdepolarizations (EADs). Our single-cell study shows that (1) the myocyte-fibroblast (MF) coupling G(j) and (2) the number N-f of fibroblasts in an MF unit lower the steepness of the APDR slope and eliminate the EAD behaviors of myocytes; we explore the pacing dependence of such EAD suppression. In our 2D simulations, we observe that a spiral-turbulence (ST) state evolves into a state with a single, rotating spiral (RS) if either (a) G(j) is large or (b) the maximum possible number of fibroblasts per myocyte N-f(max) is large. We also observe that the minimum value of G(j), for the transition from the ST to the RS state, decreases as N-f(max) increases. We find that, for the steep-APDR-induced ST state, once the MF coupling suppresses ST, the rotation period of a spiral in the RS state increases as (1) G(j) increases, with fixed N-f(max), and (2) N-f(max) increases, with fixed G(j). We obtain the boundary between ST and RS stability regions in the N-f(max)-G(j) plane. In particular, for low values of N-f(max), the value of G(j), at the ST-RS boundary, depends on the realization of the randomly distributed fibroblasts; this dependence decreases as N-f(max) increases. Our 3D studies show a similar transition from scroll-wave turbulence to a single, rotating, scroll-wave state because of the MF coupling. We examine the experimental implications of our study and propose that the suppression (a) of the steep slope of the APDR or (b) EADs can eliminate spiral-and scroll-wave turbulence in heterogeneous cardiac tissue, which has randomly distributed fibroblasts.

Bacterial growth rate and host factors as determinants of intracellular bacterial distributions in systemic Salmonella enterica infections.

Bacteria of the species Salmonella enterica cause a range of life-threatening diseases in humans and animals worldwide. The within-host quantitative, spatial, and temporal dynamics of S. enterica interactions are key to understanding how immunity acts on these infections and how bacteria evade immune surveillance. In this study, we test hypotheses generated from mathematical models of in vivo dynamics of Salmonella infections with experimental observation of bacteria at the single-cell level in infected mouse organs to improve our understanding of the dynamic interactions between host and bacterial mechanisms that determine net growth rates of S. enterica within the host. We show that both bacterial and host factors determine the numerical distributions of bacteria within host cells and thus the level of dispersiveness of the infection.

Fracture Mechanisms And Size Effects Of Brittle Metallic Foams: In Situ Compression Tests Inside Sem

In situ compressive tests on specially designed small samples made from brittle metallic foams were accomplished in a loading device equipped in the scanning electron microscopy (SEM). Each of the small samples comprises only several cells in the effective test zone (ETZ), with one major cell in the middle. In such a system one can not only obtain sequential collapse-process images of a single cell and its cell walls with high resolution, but also correlate the detailed failure behaviour of the cell walls with the stress-strain response, therefore reveal the mechanisms of energy absorption in the mesoscopic scale. Meanwhile, the stress-strain behaviour is quite different from that of bulk foams in dimensions of enough large, indicating a strong size effect. According to the in situ observations, four failure modes in the cell-wall level were summarized, and these modes account for the mesoscopic mechanisms of energy absorption. Paralleled compression tests on bulk samples were also carried out, and it is found that both fracturing of a single cell and developing of fracture bands are defect-directed or weakness-directed processes. The mechanical properties of the brittle aluminum foams obtained from the present tests agree well with the size effect model for ductile cellular solids proposed by Onck et al. (C) 2008 Elsevier Ltd. All rights reserved.

High Order Multi-Moment Constrained Finite Volume Method. Part I: Basic Formulation

A new high-order finite volume method based on local reconstruction is presented in this paper. The method, so-called the multi-moment constrained finite volume (MCV) method, uses the point values defined within single cell at equally spaced points as the model variables (or unknowns). The time evolution equations used to update the unknowns are derived from a set of constraint conditions imposed on multi kinds of moments, i.e. the cell-averaged value and the point-wise value of the state variable and its derivatives. The finite volume constraint on the cell-average guarantees the numerical conservativeness of the method. Most constraint conditions are imposed on the cell boundaries, where the numerical flux and its derivatives are solved as general Riemann problems. A multi-moment constrained Lagrange interpolation reconstruction for the demanded order of accuracy is constructed over single cell and converts the evolution equations of the moments to those of the unknowns. The presented method provides a general framework to construct efficient schemes of high orders. The basic formulations for hyperbolic conservation laws in 1- and 2D structured grids are detailed with the numerical results of widely used benchmark tests. (C) 2009 Elsevier Inc. All rights reserved.

Role of feedback and dynamics in a gene regulatory network

Cells exhibit a diverse repertoire of dynamic behaviors. These dynamic functions are implemented by circuits of interacting biomolecules. Although these regulatory networks function deterministically by executing specific programs in response to extracellular signals, molecular interactions are inherently governed by stochastic fluctuations. This molecular noise can manifest as cell-to-cell phenotypic heterogeneity in a well-mixed environment. Single-cell variability may seem like a design flaw but the coexistence of diverse phenotypes in an isogenic population of cells can also serve a biological function by increasing the probability of survival of individual cells upon an abrupt change in environmental conditions. Decades of extensive molecular and biochemical characterization have revealed the connectivity and mechanisms that constitute regulatory networks. We are now confronted with the challenge of integrating this information to link the structure of these circuits to systems-level properties such as cellular decision making. To investigate cellular decision-making, we used the well studied galactose gene-regulatory network in \textit{Saccharomyces cerevisiae}. We analyzed the mechanism and dynamics of the coexistence of two stable on and off states for pathway activity. We demonstrate that this bimodality in the pathway activity originates from two positive feedback loops that trigger bistability in the network. By measuring the dynamics of single-cells in a mixed sugar environment, we observe that the bimodality in gene expression is a transient phenomenon. Our experiments indicate that early pathway activation in a cohort of cells prior to galactose metabolism can accelerate galactose consumption and provide a transient increase in growth rate. Together these results provide important insights into strategies implemented by cells that may have been evolutionary advantageous in competitive environments.

Functional genomic studies of the structure and regulation of eukaryotic transcriptomes

The main focus of this thesis is the use of high-throughput sequencing technologies in functional genomics (in particular in the form of ChIP-seq, chromatin immunoprecipitation coupled with sequencing, and RNA-seq) and the study of the structure and regulation of transcriptomes. Some parts of it are of a more methodological nature while others describe the application of these functional genomic tools to address various biological problems. A significant part of the research presented here was conducted as part of the ENCODE (ENCyclopedia Of DNA Elements) Project.

The first part of the thesis focuses on the structure and diversity of the human transcriptome. Chapter 1 contains an analysis of the diversity of the human polyadenylated transcriptome based on RNA-seq data generated for the ENCODE Project. Chapter 2 presents a simulation-based examination of the performance of some of the most popular computational tools used to assemble and quantify transcriptomes. Chapter 3 includes a study of variation in gene expression, alternative splicing and allelic expression bias on the single-cell level and on a genome-wide scale in human lymphoblastoid cells; it also brings forward a number of critical to the practice of single-cell RNA-seq measurements methodological considerations.

The second part presents several studies applying functional genomic tools to the study of the regulatory biology of organellar genomes, primarily in mammals but also in plants. Chapter 5 contains an analysis of the occupancy of the human mitochondrial genome by TFAM, an important structural and regulatory protein in mitochondria, using ChIP-seq. In Chapter 6, the mitochondrial DNA occupancy of the TFB2M transcriptional regulator, the MTERF termination factor, and the mitochondrial RNA and DNA polymerases is characterized. Chapter 7 consists of an investigation into the curious phenomenon of the physical association of nuclear transcription factors with mitochondrial DNA, based on the diverse collections of transcription factor ChIP-seq datasets generated by the ENCODE, mouseENCODE and modENCODE consortia. In Chapter 8 this line of research is further extended to existing publicly available ChIP-seq datasets in plants and their mitochondrial and plastid genomes.

The third part is dedicated to the analytical and experimental practice of ChIP-seq. As part of the ENCODE Project, a set of metrics for assessing the quality of ChIP-seq experiments was developed, and the results of this activity are presented in Chapter 9. These metrics were later used to carry out a global analysis of ChIP-seq quality in the published literature (Chapter 10). In Chapter 11, the development and initial application of an automated robotic ChIP-seq (in which these metrics also played a major role) is presented.

The fourth part presents the results of some additional projects the author has been involved in, including the study of the role of the Piwi protein in the transcriptional regulation of transposon expression in Drosophila (Chapter 12), and the use of single-cell RNA-seq to characterize the heterogeneity of gene expression during cellular reprogramming (Chapter 13).

The last part of the thesis provides a review of the results of the ENCODE Project and the interpretation of the complexity of the biochemical activity exhibited by mammalian genomes that they have revealed (Chapters 15 and 16), an overview of the expected in the near future technical developments and their impact on the field of functional genomics (Chapter 14), and a discussion of some so far insufficiently explored research areas, the future study of which will, in the opinion of the author, provide deep insights into many fundamental but not yet completely answered questions about the transcriptional biology of eukaryotes and its regulation.

The C. elegans ALA neuron : its transcriptome and role in inducing sleep

A long-standing yet to be accomplished task in understanding behavior is to dissect the function of each gene involved in the development and function of a neuron. The C. elegans ALA neuron was chosen in this study for its known function in sleep, an ancient but less understood animal behavior. Single-cell transcriptome profiling identified 8,133 protein-coding genes in the ALA neuron, of which 57 are neuropeptide-coding genes. The most enriched genes are also neuropeptides. In combination with gain-of-function and loss-of-function assays, here I showed that the ALA-enriched FMRFamide neuropeptides, FLP-7, FLP-13, and FLP-24, are sufficient and necessary for inducing C. elegans sleep. These neuropeptides act as neuromodulators through GPCRs, NPR-7, and NPR-22. Further investigation in zebrafish indicates that FMRFamide neuropeptides are sleep-promoting molecules in animals. To correlate the behavioral outputs with genomic context, I constructed a gene regulatory network of the relevant genes controlling C. elegans sleep behavior through EGFR signaling in the ALA neuron. First, I identified an ALA cell-specific motif to conduct a genome-wide search for possible ALA-expressed genes. I then filtered out non ALA-expressed genes by comparing the motif-search genes with ALA transcriptomes from single-cell profiling. In corroborating with ChIP-seq data from modENCODE, I sorted out direct interaction of ALA-expressed transcription factors and differentiation genes in the EGFR sleep regulation pathway. This approach provides a network reference for the molecular regulation of C. elegans sleep behavior, and serves as an entry point for the understanding of functional genomics in animal behaviors.

From lakes to lungs : assessing microbial activity in diverse environments

All major geochemical cycles on the Earth’s surface are mediated by microorganisms. Our understanding of how these microbes have interacted with their environments (and vice versa) throughout Earth's history, and how they will respond to changes in the future, is primarily based on studying their activity in different environments today. The overarching questions that motivate the research presented in the two parts of this thesis -- how do microorganisms shape their environment (and vice versa)? and how can we best study microbial activity in situ? -- have arisen from the ultimate goal of being able to predict microbial activity in response to changes within their environments both past and future.

Part one focuses on work related to microbial processes in iron-rich Lake Matano and, more broadly, microbial interactions with the biogeochemical cycling of iron. Primarily, we find that the chelation of ferrous iron by organic ligands can affect the role of iron in anoxic environmental systems, enabling photomixotrophic growth of anoxygenic microorganisms with ferrous iron, as well as catalyzing the oxidation of ferrous iron by denitrification intermediates. These results imply that the ability to grow photomixotrophically on ferrous iron might be more widespread than previously assumed, and that the co-occurrence of chemical and biological processes involved in the coupled biogeochemical cycling of iron and nitrogen likely dominate organic-rich environmental systems.

Part two switches focus to in situ measurements of growth activity and comprises work related to microbial processes in the Cystic Fibrosis lung, and more broadly, the physiology of slow growth. We introduce stable isotope labeling of microbial membrane fatty acids and whole cells with heavy water as a new technique to measure microbial activity in a wide range of environments, demonstrate its application in continuous culture in the laboratory at the population and single cell level, and apply the tool to measure the in situ activity of the opportunistic pathogen Staphylococcus aureus within the environment of expectorated mucus from cystic fibrosis patients. We find that the average in situ growth rates of S. aureus fall into a range of generation times between ~12 hours and ~4 days, with substantial heterogeneity at the single-cell level. These data illustrate the use of heavy water as a universal environmental tracer for microbial activity, and highlight the crucial importance of studying the physiology of slow growth in representative laboratory systems in order to understand the role of these microorganisms in their native environments.

From molecules to organs : Microscopy and multi-scale nature of development

Morphogenesis is a phenomenon of intricate balance and dynamic interplay between processes occurring at a wide range of scales (spatial, temporal and energetic). During development, a variety of physical mechanisms are employed by tissues to simultaneously pattern, move, and differentiate based on information exchange between constituent cells, perhaps more than at any other time during an organism's life. To fully understand such events, a combined theoretical and experimental framework is required to assist in deciphering the correlations at both structural and functional levels at scales that include the intracellular and tissue levels as well as organs and organ systems. Microscopy, especially diffraction-limited light microscopy, has emerged as a central tool to capture the spatio-temporal context of life processes. Imaging has the unique advantage of watching biological events as they unfold over time at single-cell resolution in the intact animal. In this work I present a range of problems in morphogenesis, each unique in its requirements for novel quantitative imaging both in terms of the technique and analysis. Understanding the molecular basis for a developmental process involves investigating how genes and their products- mRNA and proteins-function in the context of a cell. Structural information holds the key to insights into mechanisms and imaging fixed specimens paves the first step towards deciphering gene function. The work presented in this thesis starts with the demonstration that the fluorescent signal from the challenging environment of whole-mount imaging, obtained by in situ hybridization chain reaction (HCR), scales linearly with the number of copies of target mRNA to provide quantitative sub-cellular mapping of mRNA expression within intact vertebrate embryos. The work then progresses to address aspects of imaging live embryonic development in a number of species. While processes such as avian cartilage growth require high spatial resolution and lower time resolution, dynamic events during zebrafish somitogenesis require higher time resolution to capture the protein localization as the somites mature. The requirements on imaging are even more stringent in case of the embryonic zebrafish heart that beats with a frequency of ~ 2-2.5 Hz, thereby requiring very fast imaging techniques based on two-photon light sheet microscope to capture its dynamics. In each of the hitherto-mentioned cases, ranging from the level of molecules to organs, an imaging framework is developed, both in terms of technique and analysis to allow quantitative assessment of the process in vivo. Overall the work presented in this thesis combines new quantitative tools with novel microscopy for the precise understanding of processes in embryonic development.