Notes

Influence with the Step-by-Step upon febrile children.
Circadian clocks can be found in nearly all eukaryotic organisms, as well as certain bacterial strains, including commensal microbiota. Exploring intercellular coupling among cell-autonomous circadian oscillators is crucial for understanding how cellular ensembles generate and sustain coherent circadian rhythms on the tissue level, and thus, rhythmic organ functions. Here we describe a protocol for studying intercellular coupling among peripheral circadian oscillators using three-dimensional spheroid cultures in order to measure coupling strength within peripheral clock networks. We use cell spheroids to simulate in vivo tissue integrity, as well as to increase complexity of cell-cell interactions and the abundance of potential coupling factors. Circadian rhythms are monitored using live-cell imaging of spheroids equipped with circadian reporters over several days.In mammals, molecular circadian clocks not only exist in the suprachiasmatic nucleus (SCN) but in almost all organ systems. Intriguingly, tissue clocks can operate in both isolated tissues and cell lines with endocrine signals mediating the circadian expression of local transcriptomes. This can be demonstrated by treating tissue explants with endocrine cues in a phase- and dose-dependent manner. In this chapter we provide an overview of methods to study the effects of candidate hormonal time cues on tissue clock resetting. We propose an experimental procedure based on an in vitro setup consisting of several consecutive steps in which organotypic tissue cultures or cells can be used. Our approach targets the potential resetting mechanism at three levels the hormone, the direct clock gene target, and the tissue clock response.Luciferases are a popular tool in circadian biology research as longitudinal reporters of gene expression. Here, we describe a short updated protocol for the use of an Automated Longitudinal Luciferase Imaging Gas and Temperature-Optimized Recorder (ALLIGATOR) to record cellular bioluminescence over many days. The ALLIGATOR has superior capacity and flexibility compared with traditional luminometers that employ photomultiplier tubes (PMTs), with high-throughput capability and spatial resolution. It can be readily adapted to a wide variety of applications, such as different sample types and plate sizes, under a wide range of physiologically relevant conditions.Endogenous circadian clocks play a key role in regulating a vast array of biological processes from cell cycle to metabolism, and disruption of circadian rhythms exacerbates a range of human ailments including cardiovascular, metabolic, and gastrointestinal diseases. Determining the state of a patient's circadian rhythms and clock-controlled signaling pathways has important implications for precision and personalized medicine, from improving the diagnosis of circadian-related disorders to optimizing the timing of drug delivery. Patient-derived 3-dimensional enteroids or in vitro "mini gut" is an attractive model uncovering human- and patient-specific circadian target genes that may be critical for personalized medicine. Here, we introduce several procedures to assess circadian rhythms and cell cycle dynamics in enteroids through time course sample collection methods and assay techniques including immunofluorescence, live cell confocal microscopy, and bioluminescence. These methods can be applied to evaluate the state of circadian rhythms and circadian clock-gated cell division cycles using mouse and human intestinal enteroids.There is increasing demand to control circadian clock functions in a conditional manner for deeper understanding of the circadian system as well as for potential treatment of clock-related diseases. Small-molecule compounds provide powerful tools to reveal novel functions of target proteins in the circadian clock mechanism, and can be great therapeutic candidates. Here we describe the detailed methods of measuring cellular circadian rhythms in a high-throughput manner for chemical screening to identify compounds that affect circadian rhythms by targeting clock-related proteins.Circadian rhythms are fundamental to biology and medicine and today these can be studied at the molecular level in high-throughput fashion using various omic technologies. We briefly present two resources for the study of circadian omic (e.g. transcriptomic, metabolomic, proteomic) time series. First, BIO_CYCLE is a deep-learning-based program and web server that can analyze omic time series and statistically assess their periodic nature and, when periodic, accurately infer the corresponding period, amplitude, and phase. Second, CircadiOmics is the larges annotated repository of circadian omic time series, containing over 260 experiments and 90 million individual measurements, across multiple organs and tissues, and across 9 different species. In combination, these tools enable powerful bioinformatics and systems biology analyses. The are currently being deployed in a host of different projects where they are enabling significant discoveries both tools are publicly available over the web at http//circadiomics.ics.uci.edu/ .Circadian clocks are autonomous systems able to oscillate in a self-sustained manner in the absence of external cues, although such Zeitgebers are typically present. At the cellular level, the molecular clockwork consists of a complex network of interlocked feedback loops. This chapter discusses self-sustained circadian oscillators in the context of nonlinear dynamics theory. We suggest basic steps that can help in constructing a mathematical model and introduce how self-sustained generations can be modeled using ordinary differential equations. Moreover, we discuss how coupled oscillators synchronize among themselves or entrain to periodic signals. The development of mathematical models over the last years has helped to understand such complex network systems and to highlight the basic building blocks in which oscillating systems are built upon. We argue that, through theoretical predictions, the use of simple models can guide experimental research and is thus suitable to model biological systems qualitatively.Experiments that compare rhythmic properties across different genetic alterations and entrainment conditions underlie some of the most important breakthroughs in circadian biology. A robust estimation of the rhythmic properties of the circadian signals goes hand in hand with these discoveries. Widely applied traditional signal analysis methods such as fitting cosine functions or Fourier transformations rely on the assumption that oscillation periods do not change over time. However, novel high-resolution recording techniques have shown that, most commonly, circadian signals exhibit time-dependent changes of periods and amplitudes which cannot be captured with the traditional approaches. In this chapter we introduce a method to determine time-dependent properties of oscillatory signals, using the novel open-source Python-based Biological Oscillations Analysis Toolkit (pyBOAT). We show with examples how to detect rhythms, compute and interpret high-resolution time-dependent spectral results, analyze the main oscillatory component, and to subsequently determine these main components' time-dependent instantaneous period, amplitude, and phase. We introduce step-by-step how such an analysis can be done by means of the easy-to-use point-and-click graphical user interface (GUI) provided by pyBOAT or executed within a Python programming environment. Concepts are explained using simulated signals as well as experimentally obtained time series.Circadian rhythms are part of the body's clock, which regulates several physiological and biochemical variables according to the 24-h cycle. Ample evidence indicated disturbance of the circadian clock leads to an increased susceptibility to several diseases. Therefore, a great effort has been made to find small molecules that regulate circadian rhythm by high-throughput methods. Having crystal structures of core clock proteins, makes them amenable to structure-based drug design studies. Here, we describe virtual screening methods that can be utilized for the identification of small molecules regulating the activity of core clock protein Cryptochrome 1.Human sleep is regulated by light in two fundamental ways The light-dark (LD) cycle entrains a circadian clock that in turn regulates sleep timing, and light per se can acutely inhibit sleep. Throughout evolution, these sleep regulatory systems became highly sensitive to the effects of light and they can be affected by the relatively low light intensities that are used indoors. Thus, postindustrial living conditions have created built environments that have isolated humans from the natural LD cycle and exposed them to an artificial one that can affect daily sleep timing. Studying indigenous communities that have differential access to electricity, as well as communities living in highly urbanized areas, we and others have shown that human access to artificial light has delayed the daily onset of sleep but has had a smaller effect on its offset, leading to an overall reduction in sleep duration that is pervasive in modern societies. In this chapter we discuss these studies, highlight their main findings, and point to their limitations.Attentional selection is driven, in part, by a complex interplay between endogenous and exogenous cues. Recently, one's interactions with the physical world have also been shown to bias attention. Specifically, the sense of agency that arises when our actions cause predictable outcomes biases our attention toward those things which we control. We investigated how this agency-driven attentional bias interacts with simultaneously presented endogenous (words) and exogenous (color singletons) environmental cues. Participants controlled the movement of one object while others moved independently. In a subsequent search task, targets were either the previously controlled objects or not. Targets were also validly or invalidly cued. read more Both cue types influenced attention allocation. Endogenous cues and agency-driven attentional selection were independent and additive, indicating they are separable mechanisms of selection. In contrast, exogenous cues eliminated the effects of agency, indicating that perceptually salient environmental cues can override internally derived effects of agency. This is the first demonstration of a boundary condition on agency-driven selection.In this paper we propose an anti-inertial motion (AIM) bias that can explain several intuitive physics beliefs including the straight-down belief and beliefs held concerning the pendulum problem. We show how the AIM bias also explains two new beliefs that we explore - a straight-up-and-down belief as well as a straight-out/backward bias that occurs for objects traveling in one plane that are then thrown in another plane, ostensibly affording a greater opportunity for perception of canonical motion. We then show how the AIM bias in general is invariant across perceived/imagined speed of the object carrier, only altering percentages of straight-out from backward responses, and why occluding the carrier once the object is released into a second plane does not result in more veridical perception. The AIM bias serves as a simple explanation for a family of beliefs including those in the current paper as well as those shown in previous work.
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