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Characterization of circadian systems at the organism level—a top-down approach—has led to definition of unifying properties, a hallmark of the science of chronobiology. The next challenge is to use a bottom-up approach to show how the molecular workings of the cellular circadian clock work as building blocks of those properties. We review new studies, including a recently published PLOS Biology paper by Nikhil and colleagues, that show how programmed but also stochastic generation of variation in cellular circadian period explain important adaptive features of entrained circadian phase. A recent PLOS Biology paper shows that clonal cell populations are themselves a collection heterogeneous cellular circadian clocks; this Primer explores the implications, proposing that the phase of entrainment of biological clocks (to time of day or to season) is granular, built from the contributions of individual cells. [ABSTRACT FROM AUTHOR]

Individual cells show variability in their signaling dynamics that often correlates with phenotypic responses, indicating that cell-to-cell variability is not merely noise but can have functional consequences. Based on this observation, we reasoned that cell-to-cell variability under the same treatment condition could be explained in part by a single signaling motif that maps different upstream signals into a corresponding set of downstream responses. If this assumption holds, then repeated measurements of upstream and downstream signaling dynamics in a population of cells could provide information about the underlying signaling motif for a given pathway, even when no prior knowledge of that motif exists. To test these two hypotheses, we developed a computer algorithm called MISC (Motif Inference from Single Cells) that infers the underlying signaling motif from paired time-series measurements from individual cells. When applied to measurements of transcription factor and reporter gene expression in the yeast stress response, MISC predicted signaling motifs that were consistent with previous mechanistic models of transcription. The ability to detect the underlying mechanism became less certain when a cell's upstream signal was randomly paired with another cell's downstream response, demonstrating how averaging time-series measurements across a population obscures information about the underlying signaling mechanism. In some cases, motif predictions improved as more cells were added to the analysis. These results provide evidence that mechanistic information about cellular signaling networks can be systematically extracted from the dynamical patterns of single cells. Author summary: Cells use molecular signaling networks to translate dynamically changing stimuli into appropriate downstream responses. Specialized network structures, or motifs, allow cells to properly decode a variety of temporal input signals. In this paper, we present an algorithm that infers signaling motifs from multiple examples of an upstream signal paired with its downstream response in a population of single cells. We compare the predictive power of single-cell versus averaged time-series traces and the incremental benefit of adding more single-cell traces to the algorithm. We use this approach to understand how yeast respond to environmental stresses. [ABSTRACT FROM AUTHOR]

A number of recent studies have shown that cell shape and cytoskeletal texture can be used as sensitive readouts of the physiological state of the cell. However, utilization of this information requires the development of quantitative measures that can describe relevant aspects of cell shape. In this paper we develop a toolbox, TISMorph, that calculates a set of quantitative measures to address this need. Some of the measures introduced here have been used previously, while others are new and have desirable properties for shape and texture quantification of cells. These measures, broadly classifiable into the categories of textural, irregularity and spreading measures, are tested by using them to discriminate between osteosarcoma cell lines treated with different cytoskeletal drugs. We find that even though specific classification tasks often rely on a few measures, these are not the same between all classification tasks, thus requiring the use of the entire suite of measures for classification and discrimination. We provide detailed descriptions of the measures, as well as the TISMorph package to implement them. Quantitative morphological measures that capture different aspects of cell morphology will help enhance large-scale image-based quantitative analysis, which is emerging as a new field of biological data. [ABSTRACT FROM AUTHOR]

Blocking cytokine interleukin 10 (IL-10) at the time of immunisation enhances vaccine induced T cell responses and improves control of tumour cell growth in vivo. However, the effect of an IL-10 blockade on the biological function of macrophages has not been explored. In the current paper, a macrophage precursor cell line, U937 cells, was selected to investigate the differential expression of proteins and relevant cell signalling pathway changes, when stimulated with lipopolysaccharide (LPS) in the presence of antibodies to IL-10 or IL-10 receptor. We used a quantitative proteomic strategy to investigate variations in protein profiles of U937 cells following the treatments with LPS, LPS plus human anti-IL10 antibody and anti-IL10R antibody in 24hrs, respectively. The LPS treatment significantly activated actin-related cell matrix formation and immune response pathways. The addition of anti-IL10 and anti-IL10R antibody further promoted the immune response and potentially effect macrophage survival through PI3K/AKT signalling; however, the latter appeared to also upregulated oncogene XRCC5 and Cajal body associated processes. [ABSTRACT FROM AUTHOR]

Fluorescent reporters are commonly used to quantify activities or properties of both natural and engineered cells. Fluorescence is still typically reported only in arbitrary or normalized units, however, rather than in units defined using an independent calibrant, which is problematic for scientific reproducibility and even more so when it comes to effective engineering. In this paper, we report an interlaboratory study showing that simple, low-cost unit calibration protocols can remedy this situation, producing comparable units and dramatic improvements in precision over both arbitrary and normalized units. Participants at 92 institutions around the world measured fluorescence from E. coli transformed with three engineered test plasmids, plus positive and negative controls, using simple, low-cost unit calibration protocols designed for use with a plate reader and/or flow cytometer. In addition to providing comparable units, use of an independent calibrant allows quantitative use of positive and negative controls to identify likely instances of protocol failure. The use of independent calibrants thus allows order of magnitude improvements in precision, narrowing the 95% confidence interval of measurements in our study up to 600-fold compared to normalized units. [ABSTRACT FROM AUTHOR]

With cutting-edge live microscopy and image analysis, biologists can now systematically track individual cells in complex tissues and quantify cellular behavior over extended time windows. Computational approaches that utilize the systematic and quantitative data are needed to understand how cells interact in vivo to give rise to the different cell types and 3D morphology of tissues. An agent-based, minimum descriptive modeling and analysis framework is presented in this paper to study C. elegans embryogenesis. The framework is designed to incorporate the large amounts of experimental observations on cellular behavior and reserve data structures/interfaces that allow regulatory mechanisms to be added as more insights are gained. Observed cellular behaviors are organized into lineage identity, timing and direction of cell division, and path of cell movement. The framework also includes global parameters such as the eggshell and a clock. Division and movement behaviors are driven by statistical models of the observations. Data structures/interfaces are reserved for gene list, cell-cell interaction, cell fate and landscape, and other global parameters until the descriptive model is replaced by a regulatory mechanism. This approach provides a framework to handle the ongoing experiments of single-cell analysis of complex tissues where mechanistic insights lag data collection and need to be validated on complex observations. [ABSTRACT FROM AUTHOR]

Large, naturally evolved biomolecular networks typically fulfil multiple functions. When modelling or redesigning such systems, functional subsystems are often analysed independently first, before subsequent integration into larger-scale computational models. In the design and analysis process, it is therefore important to quantitatively analyse and predict the dynamics of the interactions between integrated subsystems; in particular, how the incremental effect of integrating a subsystem into a network depends on the existing dynamics of that network. In this paper we present a framework for simulating the contribution of any given functional subsystem when integrated together with one or more other subsystems. This is achieved through a cascaded layering of a network into functional subsystems, where each layer is defined by an appropriate subset of the reactions. We exploit symmetries in our formulation to exhaustively quantify each subsystem's incremental effects with minimal computational effort. When combining subsystems, their isolated behaviour may be amplified, attenuated, or be subject to more complicated effects. We propose the concept of mutual dynamics to quantify such nonlinear phenomena, thereby defining the incompatibility and cooperativity between all pairs of subsystems when integrated into any larger network. We exemplify our theoretical framework by analysing diverse behaviours in three dynamic models of signalling and metabolic pathways: the effect of crosstalk mechanisms on the dynamics of parallel signal transduction pathways; reciprocal side-effects between several integral feedback mechanisms and the subsystems they stabilise; and consequences of nonlinear interactions between elementary flux modes in glycolysis for metabolic engineering strategies. Our analysis shows that it is not sufficient to just specify subsystems and analyse their pairwise interactions; the environment in which the interaction takes place must also be explicitly defined. Our framework provides a natural representation of nonlinear interaction phenomena, and will therefore be an important tool for modelling large-scale evolved or synthetic biomolecular networks.

Neuronal information processing is regulated by fast and localized fluctuations of brain states. Brain states reliably switch between distinct spatiotemporal signatures at a network scale even though they are composed of heterogeneous and variable rhythms at a cellular scale. We investigated the mechanisms of this network control in a conductance-based population model that reliably switches between active and oscillatory mean-fields. Robust control of the mean-field properties relies critically on a switchable negative intrinsic conductance at the cellular level. This conductance endows circuits with a shared cellular positive feedback that can switch population rhythms on and off at a cellular resolution. The switch is largely independent from other intrinsic neuronal properties, network size and synaptic connectivity. It is therefore compatible with the temporal variability and spatial heterogeneity induced by slower regulatory functions such as neuromodulation, synaptic plasticity and homeostasis. Strikingly, the required cellular mechanism is available in all cell types that possess T-type calcium channels but unavailable in computational models that neglect the slow kinetics of their activation. [ABSTRACT FROM AUTHOR]

A correction to the article "Root System Architecture from Coupling Cell Shape to Auxin Transport" published in the previous issue is presented.

Single-cell RNA-Sequencing (scRNA-seq) is the most widely used high-throughput technology to measure genome-wide gene expression at the single-cell level. One of the most common analyses of scRNA-seq data detects distinct subpopulations of cells through the use of unsupervised clustering algorithms. However, recent advances in scRNA-seq technologies result in current datasets ranging from thousands to millions of cells. Popular clustering algorithms, such as k-means, typically require the data to be loaded entirely into memory and therefore can be slow or impossible to run with large datasets. To address this problem, we developed the mbkmeans R/Bioconductor package, an open-source implementation of the mini-batch k-means algorithm. Our package allows for on-disk data representations, such as the common HDF5 file format widely used for single-cell data, that do not require all the data to be loaded into memory at one time. We demonstrate the performance of the mbkmeans package using large datasets, including one with 1.3 million cells. We also highlight and compare the computing performance of mbkmeans against the standard implementation of k-means and other popular single-cell clustering methods. Our software package is available in Bioconductor at https://bioconductor.org/packages/mbkmeans. Author summary: We developed the mbkmeans package (https://bioconductor.org/packages/mbkmeans) in Bioconductor, an open-source implementation of the mini-batch k-means algorithm. Our package allows for on-disk data representations, such as the common HDF5 file format widely used for single-cell data, that do not require all the data to be loaded into memory at one time. [ABSTRACT FROM AUTHOR]

Uptake of modified lipoproteins by macrophages turns them into foam cells, the hallmark of the atherosclerotic plaque. The initiation and progression of atherosclerosis have been associated with mitochondrial dysfunction. It is known that aggregated low-density lipoproteins (agLDL) induce massive cholesterol accumulation in macrophages in contrast with native LDL (nLDL) and oxidized LDL (oxLDL). In the present study we aimed to assess the effect of agLDL on the mitochondria and ER function in macrophage-derived foam cells, in an attempt to estimate the potential of these cells, known constituents of early fatty streaks, to generate atheroma in the absence of oxidative stress. Results show that agLDL induce excessive accumulation of free (FC) and esterified cholesterol in THP-1 macrophages and determine mitochondrial dysfunction expressed as decreased mitochondrial membrane potential and diminished intracellular ATP levels, without generating mitochondrial reactive oxygen species (ROS) production. AgLDL did not stimulate intracellular ROS (superoxide anion or hydrogen peroxide) production, and did not trigger endoplasmic reticulum stress (ERS) or apoptosis. In contrast to agLDL, oxLDL did not modify FC levels, but stimulated the accumulation of 7-ketocholesterol in the cells, generating oxidative stress which is associated with an increased mitochondrial dysfunction, ERS and apoptosis. Taken together, our results reveal that agLDL induce foam cells formation and mild mitochondrial dysfunction in human macrophages without triggering oxidative or ERS. These data could partially explain the early formation of fatty streaks in the intima of human arteries by interaction of monocyte-derived macrophages with non-oxidatively aggregated LDL generating foam cells, which cannot evolve into atherosclerotic plaques in the absence of the oxidative stress. [ABSTRACT FROM AUTHOR]

The ability of butyrate to promote differentiation of cancer cells has important implication for colorectal cancer (CRC) prevention and therapy. In this study, we examined the effect of sodium butyrate (NaBT) on the energy metabolism of colon adenocarcinoma Caco-2 cells coupled with their differentiation. NaBT increased the activity of alkaline phosphatase indicating differentiation of Caco-2 cells. Changes in the expression of pluripotency-associated markers OCT4, NANOG and SOX2 were characterized during the induced differentiation at mRNA level along with the measures that allowed distinguishing the expression of different transcript variants. The functional activity of mitochondria was studied by high-resolution respirometry. Glycolytic pathway and phosphotransfer network were analyzed using enzymatical assays. The treatment of Caco-2 cells with NaBT increased production of ATP by oxidative phosphorylation, enhanced mitochondrial spare respiratory capacity and caused rearrangement of the cellular phosphotransfer networks. The flexibility of phosphotransfer networks depended on the availability of glutamine, but not glucose in the cell growth medium. These changes were accompanied by suppressed cell proliferation and altered gene expression of the main pluripotency-associated transcription factors. This study supports the view that modulating cell metabolism through NaBT can be an effective strategy for treating CRC. Our data indicate a close relationship between the phosphotransfer performance and metabolic plasticity of CRC, which is associated with the cell differentiation state. [ABSTRACT FROM AUTHOR]

The Golgi cells are the main inhibitory interneurons of the cerebellar granular layer. Although recent works have highlighted the complexity of their dendritic organization and synaptic inputs, the mechanisms through which these neurons integrate complex input patterns remained unknown. Here we have used 8 detailed morphological reconstructions to develop multicompartmental models of Golgi cells, in which Na, Ca, and K channels were distributed along dendrites, soma, axonal initial segment and axon. The models faithfully reproduced a rich pattern of electrophysiological and pharmacological properties and predicted the operating mechanisms of these neurons. Basal dendrites turned out to be more tightly electrically coupled to the axon initial segment than apical dendrites. During synaptic transmission, parallel fibers caused slow Ca-dependent depolarizations in apical dendrites that boosted the axon initial segment encoder and Na-spike backpropagation into basal dendrites, while inhibitory synapses effectively shunted backpropagating currents. This oriented dendritic processing set up a coincidence detector controlling voltage-dependent NMDA receptor unblock in basal dendrites, which, by regulating local calcium influx, may provide the basis for spike-timing dependent plasticity anticipated by theory. Author summary: The Golgi cells are the main inhibitory interneurons of the cerebellum granular layer and play a fundamental role in controlling cerebellar processing. However, it was unclear how spikes are processed in the dendrites by specific sets of ionic channels and how they might contribute to integrate synaptic inputs and plasticity. Here we have developed detailed multicompartmental models of Golgi cells that faithfully reproduced a large set of experimental findings and revealed the nature of signal interchange between dendrites and axo-somatic compartments. A main prediction of the models is that synaptic activation of apical dendrites can effectively trigger spike generation in the axonal initial segment followed by rapid spike backpropagation into basal dendrites. Here, incoming mossy fiber inputs and backpropagating spikes regulate the voltage-dependent unblock of NMDA channels and the induction of spike timing-dependent plasticity (STDP). STDP, which was predicted by theory, may therefore be controlled by contextual information provided by parallel fibers and integrated in apical dendrites, supporting the view that spike timing is fundamental to control synaptic plasticity at the cerebellar input stage. [ABSTRACT FROM AUTHOR]

Cerebellar stellate cells (CSCs) are spontaneously active, tonically firing (5-30 Hz), inhibitory interneurons that synapse onto Purkinje cells. We previously analyzed the excitability properties of CSCs, focusing on four key features: type I excitability, non-monotonic first-spike latency, switching in responsiveness and runup (i.e., temporal increase in excitability during whole-cell configuration). In this study, we extend this analysis by using whole-cell configuration to show that these neurons can also burst when treated with certain pharmacological agents separately or jointly. Indeed, treatment with (4-Aminopyridine) 4-AP, a partial blocker of delayed rectifier and A-type K+ channels, at low doses induces a bursting profile in CSCs significantly different than that produced at high doses or when it is applied at low doses but with cadmium (Cd2+), a blocker of high voltage-activated (HVA) Ca2+ channels. By expanding a previously revised Hodgkin–Huxley type model, through the inclusion of Ca2+-activated K+ (K(Ca)) and HVA currents, we explain how these bursts are generated and what their underlying dynamics are. Specifically, we demonstrate that the expanded model preserves the four excitability features of CSCs, as well as captures their bursting patterns induced by 4-AP and Cd2+. Model investigation reveals that 4-AP is potentiating HVA, inducing square-wave bursting at low doses and pseudo-plateau bursting at high doses, whereas Cd2+ is potentiating K(Ca), inducing pseudo-plateau bursting when applied in combination with low doses of 4-AP. Using bifurcation analysis, we show that spike adding in square-wave bursts is non-sequential when gradually changing HVA and K(Ca) maximum conductances, delayed Hopf is responsible for generating the plateau segment within the active phase of pseudo-plateau bursts, and bursting can become "chaotic" when HVA and K(Ca) maximum conductances are made low and high, respectively. These results highlight the secondary effects of the drugs applied and suggest that CSCs have all the ingredients needed for bursting. Author summary: Excitable cells, including neurons, fire action potentials (APs) in their membrane voltage that allow them to communicate with each other and to serve certain physiological purposes. They do so either tonically by firing APs periodically, or episodically by repeatedly firing clusters of APs (called bursts) separated by quiescent periods. Each one of those firing patterns can be neuron-specific and dependent on synaptic inputs and/or their physiological environment. Cerebellar stellate cells (CSCs) that synapse onto Purkinje cells, the sole output of the cerebellum responsible for motor control, are spontaneously active inhibitory interneurons that fire APs tonically. We previously studied the excitability properties of these neurons and showed that they possess several important key features, including type I excitability, runup, non-monotonic first spike latency and switching in responsiveness. In this study, we show that CSCs can also exhibit two modes of burst firing, called square-wave and pseudo-plateau, when treated with certain pharmacological agents. Using bifurcation theory, we demonstrate that spike adding in the square-wave burst is non-sequential, changing by several spikes when certain conductances are altered gradually. This study thus sheds lights onto the overall effects of the pharmacological agents and highlights the ability of CSCs to burst in certain biological conditions. [ABSTRACT FROM AUTHOR]

Changes in cell identities and positions underlie tissue development and disease progression. Although single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRINSHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity, and quantitative qualities of SCRINSHOT facilitate single-cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions. This study presents SCRINSHOT, an amenable, multiplex RNA-mapping method, applicable to a wide variety of tissue types and conditions. It can function quantitatively across a broad range of expression levels and detect even rare cell types, facilitating the creation of digital tissue maps with single-cell resolution. [ABSTRACT FROM AUTHOR]

The fate of the ICM in humans is still unknown, due to the ethical difficulties surrounding experimentation in this field. In this study we have explored the existing time-lapse recording data of embryos in the early stages of development, taking advantage of the large refractile bodies (RBs) within blastomeres as cellular markers. Our study found that the cellular composition of the ICM in humans is largely determined at the time of the fourth division and blastomeres which cleave first to fourth, during the fourth division from 8 cells to 16 cells, have the potential to be incorporated in the ICM. [ABSTRACT FROM AUTHOR]

Plant cells undergo two types of cell cycles–the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2'-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Comparison of the sequence-based RT profiles showed that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle and the fact that endocycling is typically associated with cell differentiation. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small with a median size of 135 kb, shift to a later RT in the endocycle, and are enriched for genes expressed in the root tip. We found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere, which ranged between 1 and 2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells but primarily in late S phase of the endocycle. In contrast, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in 8C vs 2C nuclei suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and possible reduction in CENH3 enrichment after endocycle replication is consistent with a hypothesis that centromeres are inactivated when their function is no longer needed. Author summary: In traditional cell division, or mitosis, a cell's genetic material is duplicated and then split between two daughter cells. In contrast, in some specialized cell types, the DNA is duplicated a second time without an intervening division step, resulting in cells that carry twice as much DNA. This phenomenon, which is called the endocycle, is common during plant development. At each step, DNA replication follows an ordered program in which highly compacted DNA is unraveled and replicated in sections at different times during the synthesis (S) phase. In plants, it is unclear whether traditional and endocycle programs are the same, especially since endocycling cells are typically in the process of differentiation. Using root tips of maize, we found that in comparison to replication in the mitotic cell cycle, there is a small portion of the genome whose replication in the endocycle is shifted in time, usually to later in S phase. Some of these regions are scattered around the genome and mostly coincide with active genes. However, the most prominent shifts occur in centromeres. The shift to later replication in centromeres is noteworthy because they orchestrate the process of separating duplicated chromosomes into daughter cells, a function that is not needed in the endocycle. [ABSTRACT FROM AUTHOR]

We present VALERIE (Visualising alternative splicing events from single-cell ribonucleic acid-sequencing experiments), an R package for visualising alternative splicing events at single-cell resolution. To explore any given specified genomic region, corresponding to an alternative splicing event, VALERIE generates an ensemble of informative plots to visualise cell-to-cell heterogeneity of alternative splicing profiles across single cells and performs statistical tests to compare percent spliced-in (PSI) values across the user-defined groups of cells. Among the features available, VALERIE displays PSI values, in lieu of read coverage, which is more suitable for representing alternative splicing profiles for a large number of samples typically generated by single-cell RNA-sequencing experiments. VALERIE is available on the Comprehensive R Archive Network (CRAN): https://cran.r-project.org/web/packages/VALERIE/index.html. [ABSTRACT FROM AUTHOR]

The correct statement is: All RNA sequence data from this study have been submitted to NCBI (https://www.ncbi.nlm.nih.gov/geo) under accession number GSE118549. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012570 http://proteomecentral.proteomexchange.org/cgi/GetDataset. [Extracted from the article]

Various nanopatterning techniques have been developed to improve cell proliferation and differentiation efficiency. As we previously reported, nanopillars and pores are able to sustain human pluripotent stem cells and differentiate pancreatic cells. From this, the nanoscale patterns would be effective environment for the co-culturing of epithelial and mesenchymal cell types. Interestingly, the nanopatterning selectively reduced the proliferative rate of mesenchymal cells while increasing the expression of adhesion protein in epithelial type cells. Additionally, co-cultured cells on the nanopatterning were not negatively affected in terms of cell function metabolic ability or cell survival. This is in contrast to conventional co-culturing methods such as ultraviolet or chemical treatments. The nanopatterning appears to be an effective environment for mesenchymal co-cultures with typically low proliferative rates cells such as astrocytes, neurons, melanocytes, and fibroblasts without using potentially damaging treatments. [ABSTRACT FROM AUTHOR]

Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in the regulation of cell proliferation and differentiation. TAZ activity changes in response to the cellular environment such as mechanic and nutritional stimuli, osmolarity, and hypoxia. To understand the physiological roles of TAZ, chemical compounds that activate TAZ in cells are useful as experimental reagents. Kaempferol, TM-25659, and ethacridine are reported as TAZ activators. However, as each TAZ activator has a distinct property in cellular functions, additional TAZ activators are awaiting. We screened for TAZ activators and previously reported IB008738 as a TAZ activator that promotes myogenesis in C2C12 cells. In this study, we have characterized IBS004735 that was obtained in the same screening. IBS004735 also promotes myogenesis in C2C12 cells, but is not similar to IBS008738 in the structure. IBS004735 activates TAZ via Akt and has no effect on TAZ phosphorylation, which is the well-described key modification to regulate TAZ activity. Thus, we introduce IBS004735 as a novel TAZ activator that regulates TAZ in a yet unidentified mechanism. [ABSTRACT FROM AUTHOR]

Human epidemiological studies have shown that paternal aging as one of the risk factors for neurodevelopmental disorders, such as autism, in offspring. A recent study has suggested that factors other than de novo mutations due to aging can influence the biology of offspring. Here, we focused on epigenetic alterations in sperm that can influence developmental programs in offspring. In this study, we qualitatively and semiquantitatively evaluated histone modification patterns in male germline cells throughout spermatogenesis based on immunostaining of testes taken from young (3 months old) and aged (12 months old) mice. Although localization patterns were not obviously changed between young and aged testes, some histone modification showed differences in their intensity. Among histone modifications that repress gene expression, histone H3 lysine 9 trimethylation (H3K9me3) was decreased in the male germline cells of the aged testis, while H3K27me2/3 was increased. The intensity of H3K27 acetylation (ac), an active mark, was lower/higher depending on the stages in the aged testis. Interestingly, H3K27ac was detected on the putative sex chromosomes of round spermatids, while other chromosomes were occupied by a repressive mark, H3K27me3. Among other histone modifications that activate gene expression, H3K4me2 was drastically decreased in the male germline cells of the aged testis. In contrast, H3K79me3 was increased in M-phase spermatocytes, where it accumulates on the sex chromosomes. Therefore, aging induced alterations in the amount of histone modifications and in the differences of patterns for each modification. Moreover, histone modifications on the sex chromosomes and on other chromosomes seems to be differentially regulated by aging. These findings will help elucidate the epigenetic mechanisms underlying the influence of paternal aging on offspring development. [ABSTRACT FROM AUTHOR]

Autotaxin (ATX) is a secreted lysophospholipase D catalyzing the extracellular production of lysophosphatidic acid (LPA), a growth factor-like signaling lysophospholipid. ATX and LPA signaling have been incriminated in the pathogenesis of different chronic inflammatory diseases and various types of cancer. In this report, deregulated ATX and LPA levels were detected in the spinal cord and plasma of mice during the development of experimental autoimmune encephalomyelitis (EAE). Among the different sources of ATX expression in the inflamed spinal cord, F4/80+ CD11b+ cells, mostly activated macrophages and microglia, were found to express ATX, further suggesting an autocrine role for ATX/LPA in their activation, an EAE hallmark. Accordingly, ATX genetic deletion from CD11b+ cells attenuated the severity of EAE, thus proposing a pathogenic role for the ATX/LPA axis in neuroinflammatory disorders. [ABSTRACT FROM AUTHOR]

Background and aims: Radiotherapy is one of the major remedies for the treatment of cancer, including nasopharyngeal carcinoma (NPC). Radioresistance occurs very often in target cells that is a large drawback in cancer treated with radiotherapy. Livin involves the over-growth of cancer cells. This study aims to investigate the role of livin in the radioresistance formation in NPC cells. Methods: NPC cell lines were exposed to small doses of irradiation to establish a cell model of radioresistance, in which the role of livin in the development of radioresistance was evaluated. Results: The expression of livin was observed in NPC cells, which was significantly increased after exposing to small doses of irradiation. A negative correlation was detected between livin and Fas expression in NPC cells. Livin formed a complex with heat shock factor-1 (HSF1, the transcription factor of Fas) in NPC cells after irradiation, which sped up ubiquitination of HSF1. Livin was involved in suppressing Fas expression in NPC cells with radioresistance. Exposure to livin inhibitors prevented radioresistance development and overcame the established radioresistance in NPC cells. Conclusions: Livin expression in NPC cells plays a critical role in the development of radioresistance. Depletion of livin increases the sensitiveness of NPC cells to irradiation. Target therapy against livin may have the translational potential for the treatment of NPC. [ABSTRACT FROM AUTHOR]

Habituation, defined as the reversible decrement of a response during repetitive stimulation, is widely established as a form of non-associative learning. Though more commonly ascribed to neural cells and systems, habituation has also been observed in single aneural cells, although evidence is limited. Considering the generalizability of the habituation process, we tested the degree to which organism-level behavioral and single cell manifestations were similar. Human embryonic kidney (HEK) cells that overexpressed an optogenetic actuator were photostimulated to test the effect of different stimulation protocols on cell responses. Depolarization induced by the photocurrent decreased successively over the stimulation protocol and the effect was reversible upon withdrawal of the stimulus. In addition to frequency- and intensity-dependent effects, the history of stimulations on the cells impacted subsequent depolarization in response to further stimulation. We identified tetraethylammonium (TEA)-sensitive native K+ channels as one of the mediators of this habituation phenotype. Finally, we used a theoretical model of habituation to elucidate some mechanistic aspects of the habituation response. In conclusion, we affirm that habituation is a time- and state-dependent biological strategy that can be adopted also by individual non-neuronal cells in response to repetitive stimuli. [ABSTRACT FROM AUTHOR]

Descending cholinergic fibers innervate the cochlear nucleus. Spherical bushy cells, principal neurons of the anterior part of the ventral cochlear nucleus, are depolarized by cholinergic agonists on two different time scales. A fast and transient response is mediated by alpha-7 homomeric nicotinic receptors while a slow and long-lasting response is mediated by muscarinic receptors. Spherical bushy cells were shown to express M3 receptors, but the receptor subtypes involved in the slow muscarinic response were not physiologically identified yet. Whole-cell patch clamp recordings combined with pharmacology and immunohistochemistry were performed to identify the muscarinic receptor subtypes and the effector currents involved. Spherical bushy cells also expressed both M1 and M2 receptors. The M1 signal was stronger and mainly somatic while the M2 signal was localized in the neuropil and on the soma of bushy cells. Physiologically, the M-current was observed for the gerbil spherical bushy cells and was inhibited by oxotremorine-M application. Surprisingly, long application of carbachol showed only a transient depolarization. Even though no muscarinic depolarization could be detected, the input resistance increased suggesting a decrease in the cell conductance that matched with the closure of M-channels. The hyperpolarization-activated currents were also affected by muscarinic activation and counteracted the effect of the inactivation of M-current on the membrane potential. We hypothesize that this double muscarinic action might allow adaptation of effects during long durations of cholinergic activation. [ABSTRACT FROM AUTHOR]

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) has revolutionized our understanding of chromatin-related biological processes. The method, however, requires thousands of cells and has therefore limited applications in situations where cell numbers are limited. Here we describe a novel method called Restriction Assisted Tagmentation Chromatin Immunoprecipitation (RAT-ChIP) that enables global histone modification profiling from as few as 100 cells. The method is simple, cost-effective and takes a single day to complete. We demonstrate the sensitivity of the method by deriving the first genome-wide maps of histone H3K4me3 and H3K27me3 modifications of inner cell mass and trophectoderm of bovine blastocyst stage embryos. [ABSTRACT FROM AUTHOR]

Immune cell infiltration of tumors and the tumor microenvironment can be an important component for determining patient outcomes. For example, immune and stromal cell presence inferred by deconvolving patient gene expression data may help identify high risk patients or suggest a course of treatment. One particularly powerful family of deconvolution techniques uses signature matrices of genes that uniquely identify each cell type as determined from single cell type purified gene expression data. Many methods from this family have been recently published, often including new signature matrices appropriate for a single purpose, such as investigating a specific type of tumor. The package ADAPTS helps users make the most of this expanding knowledge base by introducing a framework for cell type deconvolution. ADAPTS implements modular tools for customizing signature matrices for new tissue types by adding custom cell types or building new matrices de novo, including from single cell RNAseq data. It includes a common interface to several popular deconvolution algorithms that use a signature matrix to estimate the proportion of cell types present in heterogenous samples. ADAPTS also implements a novel method for clustering cell types into groups that are difficult to distinguish by deconvolution and then re-splitting those clusters using hierarchical deconvolution. We demonstrate that the techniques implemented in ADAPTS improve the ability to reconstruct the cell types present in a single cell RNAseq data set in a blind predictive analysis. ADAPTS is currently available for use in R on CRAN and GitHub. [ABSTRACT FROM AUTHOR]

Single-cell RNA-sequencing data generated by a variety of technologies, such as Drop-seq and SMART-seq, can reveal simultaneously the mRNA transcript levels of thousands of genes in thousands of cells. It is often important to identify informative genes or cell-type-discriminative markers to reduce dimensionality and achieve informative cell typing results. We present an ab initio method that performs unsupervised marker selection by identifying genes that have subpopulation-discriminative expression levels and are co- or mutually-exclusively expressed with other genes. Consistent improvements in cell-type classification and biologically meaningful marker selection are achieved by applying SCMarker on various datasets in multiple tissue types, followed by a variety of clustering algorithms. The source code of SCMarker is publicly available at https://github.com/KChen-lab/SCMarker. Single cell RNA-sequencing technology simultaneously provides the mRNA transcript levels of thousands of genes in thousands of cells. A frequent requirement of single cell expression analysis is the identification of markers which may explain complex cellular states or tissue composition. We propose a new marker selection strategy (SCMarker) to accurately delineate cell types in single cell RNA-sequencing data by identifying genes that have bi/multi-modally distributed expression levels and are co- or mutually-exclusively expressed with some other genes. Our method can determine the cell-type-discriminative markers without referencing to any known transcriptomic profiles or cell ontologies, and consistently achieves accurate cell-type-discriminative marker identification in a variety of scRNA-seq datasets. [ABSTRACT FROM AUTHOR]

Transforming growth factor‑β1 (TGF-β1) is a versatile cytokine. It has context-dependent pro- and anti-cell proliferation functions. Activation of latent TGF-β1 requires release of the growth factor from pro-complexes and is regulated through TGF-β binding proteins. Two types of TGF-β binding partners, latent TGF-β-binding proteins (LTBPs) and leucine-rich-repeat-containing protein 32 (LRRC32), have been identified and their expression are cell specific. TGF-β1 also plays important roles in acute myeloid leukemia (AML) cells. However, the expression of LTBPs and LRRC32 are lacking in myeloid lineage cells and the binding protein of TGF-β1 in these cells are unknown. Here we show that a novel leucine-rich-repeat-containing protein family member, LRRC33, with high mRNA level in AML cells, to be the binding and regulating protein of TGF-β1 in AML cells. Using two representative cell lines MV4-11 and AML193, we demonstrate that the protein expression of LRRC33 and TGF-β1 are correlated. LRRC33 co-localizes and forms complex with latent TGF-β1 protein on the cell surface and intracellularly in these cells. Similar as in other cell types, the activation of TGF-β1 in MV4-11 and AML193 cells are also integrin dependent. We anticipate our study to be a starting point of more comprehensive research on LRRC33 as novel TGF-β regulating protein and potential non-genomic based drug target for AML and other myeloid malignancy. [ABSTRACT FROM AUTHOR]

CD34+ cells maintain vascular homeostasis and predict cardiovascular outcomes. We previously evaluated the association of CD34+ cells with cardiovascular disease (CVD) events over 23 months, but long-term CVD outcomes in relation to levels of CD34+ cells in patients on maintenance hemodialysis are unclear. Herein, we analyzed the long-term predictive potential levels of CD34+ cells for CVD outcomes and all-cause mortality. Between March 2005 and May 2005, we enrolled 215 patients on maintenance hemodialysis at Nagoya Kyoritsu Hospital and followed them up to 12.8 years. According to the CD34+ cell counts, patients were classified into the lowest, medium, and highest tertiles. Levels of CD34+ cells were analyzed in association with four-point major adverse CV events (MACEs), CVD death, and all-cause mortality. In univariate analysis age, smoking habit, lower geriatric nutrition risk index, lower calcium × phosphate product, and lower intact parathyroid hormone were significantly associated with the lowest tertile. Whereas, in multivariate analysis, age and smoking habit were significantly associated with the lowest tertile. Among 139 (64.7%) patients who died during a mean follow-up period of 8.0 years, 39 (28.1%) patients died from CVD. Patients in the lowest tertile had a significantly lower survival rate than those in the medium and highest tertiles (p ≤ 0.001). Using multivariable analyses, the lowest tertile was significantly associated with four-point MACEs (hazard ratio 1.80, p = 0.023) and CVD death (hazard ratio 2.50, p = 0.011). In conclusion, our long-term observational study revealed that a low level of CD34+ cells in the circulation predicts CVD outcomes among patients on maintenance hemodialysis. [ABSTRACT FROM AUTHOR]

A physiologically relevant in vitro human-based model could be the ‘gold standard’ to clarify the pathological steps involved in glaucoma onset. In this regard, human 3D cultures may represent an excellent starting point to achieve this goal. Indeed, the 3D matrix allows to re-create the in vivo-like tissue architecture, maintaining its functionality and cellular behaviour, compared to the 2D model. Thus, we propose a comparison between the 2D and 3D in vitro models of human trabecular meshwork cells in terms of cellular responses after chronic stress exposure. Our results showed that 3D-cells are more sensitive to intracellular reactive oxidative specie production induced by hydrogen peroxide treatment, compared to 2D cultures. Additionally, in 3D cultures a more accurate regulation of the apoptosis trigger and cell adaptation mechanisms was detected than in 2D models. In line with these findings, the 3D-HTMC model shows the ability to better mimic the in vivo cell behaviour in adaptive responses to chronic oxidative stress than 2D. [ABSTRACT FROM AUTHOR]

Cord blood (CB) mononuclear cells (MNC) are being tested in clinical trials to treat hypoxic-ischemic (HI) brain injuries. Although early results are encouraging, mechanisms underlying potential clinical benefits are not well understood. To explore these mechanisms further, we exposed mouse brain organotypic slice cultures to oxygen and glucose deprivation (OGD) and then treated the brain slices with cells from CB or adult peripheral blood (PB). We found that CB-MNCs protect neurons from OGD-induced death and reduced both microglial and astrocyte activation. PB-MNC failed to affect either outcome. The protective activities were largely mediated by factors secreted by CB-MNC, as direct cell-to-cell contact between the injured brain slices and CB cells was not essential. To determine if a specific subpopulation of CB-MNC are responsible for these protective activities, we depleted CB-MNC of various cell types and found that only removal of CB CD14+ monocytes abolished neuroprotection. We also used positively selected subpopulations of CB-MNC and PB-MNC in this assay and demonstrated that purified CB-CD14+ cells, but not CB-PB CD14+ cells, efficiently protected neuronal cells from death and reduced glial activation following OGD. Gene expression microarray analysis demonstrated that compared to PB-CD14+ monocytes, CB-CD14+ monocytes over-expressed several secreted proteins with potential to protect neurons. Differential expression of five candidate effector molecules, chitinase 3-like protein-1, inhibin-A, interleukin-10, matrix metalloproteinase-9 and thrombospondin-1, were confirmed by western blotting, and immunofluorescence. These findings suggest that CD14+ monocytes are a critical cell-type when treating HI with CB-MNC. [ABSTRACT FROM AUTHOR]

Spontaneous emergence of synchronized population activity is a characteristic feature of developing brain circuits. Recent experiments in the developing neo-cortex showed the existence of driver cells able to impact the synchronization dynamics when single-handedly stimulated. We have developed a spiking network model capable to reproduce the experimental results, thus identifying two classes of driver cells: functional hubs and low functionally connected (LC) neurons. The functional hubs arranged in a clique orchestrated the synchronization build-up, while the LC drivers were lately or not at all recruited in the synchronization process. Notwithstanding, they were able to alter the network state when stimulated by modifying the temporal activation of the functional clique or even its composition. LC drivers can lead either to higher population synchrony or even to the arrest of population dynamics, upon stimulation. Noticeably, some LC driver can display both effects depending on the received stimulus. We show that in the model the presence of inhibitory neurons together with the assumption that younger cells are more excitable and less connected is crucial for the emergence of LC drivers. These results provide a further understanding of the structural-functional mechanisms underlying synchronized firings in developing circuits possibly related to the coordinated activity of cell assemblies in the adult brain. [ABSTRACT FROM AUTHOR]

The cure rate for late stage epithelial ovarian cancer (EOC) has not significantly improved over several decades. New and more effective targets and treatment modalities are urgently needed. RNA-seq analyses of a syngeneic EOC cell pair, representing more and less aggressive tumor cells in vivo were conducted. Bioinformatics analyses of the RNA-seq data and biological signaling and function studies have identified new targets, such as ZIP4 in EOC. Many up-regulated tumor promoting signaling pathways have been identified which are mainly grouped into three cellular activities: 1) cell proliferation and apoptosis resistance; 2) cell skeleton and adhesion changes; and 3) carbohydrate metabolic reprograming. Unexpectedly, lipid metabolism has been the major down-regulated signaling pathway in the more aggressive EOC cells. In addition, we found that hypoxic responsive genes were at the center stage of regulation and detected functional changes were related to cancer stem cell-like activities. Moreover, our genetic, cellular, biochemical, and lipidomic analyses indicated that cells grown in 2D vs. 3D, or attached vs. suspended had dramatic changes. The important clinical implications of peritoneal cavity floating tumor cells are supported by the data proved in this work. Overall, the RNA-seq data provide a landscape of gene expression alterations during tumor progression. [ABSTRACT FROM AUTHOR]

A major challenge in systems biology is to infer the parameters of regulatory networks that operate in a noisy environment, such as in a single cell. In a stochastic regime it is hard to distinguish noise from the real signal and to infer the noise contribution to the dynamical behavior. When the genetic network displays oscillatory dynamics, it is even harder to infer the parameters that produce the oscillations. To address this issue we introduce a new estimation method built on a combination of stochastic simulations, mass action kinetics and ensemble network simulations in which we match the average periodogram and phase of the model to that of the data. The method is relatively fast (compared to Metropolis-Hastings Monte Carlo Methods), easy to parallelize, applicable to large oscillatory networks and large (~2000 cells) single cell expression data sets, and it quantifies the noise impact on the observed dynamics. Standard errors of estimated rate coefficients are typically two orders of magnitude smaller than the mean from single cell experiments with on the order of ~1000 cells. We also provide a method to assess the goodness of fit of the stochastic network using the Hilbert phase of single cells. An analysis of phase departures from the null model with no communication between cells is consistent with a hypothesis of Stochastic Resonance describing single cell oscillators. Stochastic Resonance provides a physical mechanism whereby intracellular noise plays a positive role in establishing oscillatory behavior, but may require model parameters, such as rate coefficients, that differ substantially from those extracted at the macroscopic level from measurements on populations of millions of communicating, synchronized cells. [ABSTRACT FROM AUTHOR]

Cell size is thought to play an important role in choosing between temporal and spatial sensing in chemotaxis. Large cells are thought to use spatial sensing due to large chemical difference at its ends whereas small cells are incapable of spatial sensing due to rapid homogenization of proteins within the cell. However, small cells have been found to polarize and large cells like sperm cells undergo temporal sensing. Thus, it remains an open question what exactly governs spatial versus temporal sensing. Here, we identify the factors that determines sensing choices through mathematical modeling of chemotactic circuits. Comprehensive computational search of three-node signaling circuits has identified the negative integral feedback (NFB) and incoherent feedforward (IFF) circuits as capable of adaptation, an important property for chemotaxis. Cells are modeled as one-dimensional circular system consisting of diffusible activator, inactivator and output proteins, traveling across a chemical gradient. From our simulations, we find that sensing outcomes are similar for NFB or IFF circuits. Rather than cell size, the relevant parameters are the 1) ratio of cell speed to the product of cell diameter and rate of signaling, 2) diffusivity of the output protein and 3) ratio of the diffusivities of the activator to inactivator protein. Spatial sensing is favored when all three parameters are low. This corresponds to a cell moving slower than the time it takes for signaling to propagate across the cell diameter, has an output protein that is polarizable and has a local-excitation global-inhibition system to amplify the chemical gradient. Temporal sensing is favored otherwise. We also find that temporal sensing is more robust to noise. By performing extensive literature search, we find that our prediction agrees with observation in a wide range of species and cell types ranging from E. coli to human Fibroblast cells and propose that our result is universally applicable. [ABSTRACT FROM AUTHOR]

Experimentally derived apparent permeabilities, Papp, through cell monolayers such as Caco-2 and MDCK are considered to be an in-vitro gold standard for assessing the uptake efficiency of drugs. Here, we present a mechanistic model that describes ‘passive’ Papp values (i.e., neglecting active transport) by accounting for the different resistances solutes encounter when permeating a cell monolayer. We described three parallel permeation pathways, namely a cytosolic-, paracellular-, and lateral route, each of which consists of a number of serial resistances. These resistances were accounted for via a mechanistic depiction of the underlying processes that are largely based on literature work. For the present Papp dataset, about as much chemicals are dominated by the cytosolic route as were dominated by the paracellular route, while the lateral route was negligible. For the cytosolic route by far the most chemicals found their main resistance in the various water layers and not in the membrane. Although correlations within the subclasses of chemicals dominated by a specific permeation route were rather poor, we could overall satisfyingly predict Papp for 151 chemicals at a pH of 7.4 (R2 = 0.77, RMSE = 0.48). For a specific evaluation of the intrinsic membrane permeability, Pm, a second experimental dataset based on experiments with black lipid membranes, BLM, was evaluated. Pm could be predicted for 37 chemicals with R2 = 0.91 and RMSE = 0.64 log units. [ABSTRACT FROM AUTHOR]

The mean conditional fixation time of a mutant is an important measure of stochastic population dynamics, widely studied in ecology and evolution. Here, we investigate the effect of spatial randomness on the mean conditional fixation time of mutants in a constant population of cells, N. Specifically, we assume that fitness values of wild type cells and mutants at different locations come from given probability distributions and do not change in time. We study spatial arrangements of cells on regular graphs with different degrees, from the circle to the complete graph, and vary assumptions on the fitness probability distributions. Some examples include: identical probability distributions for wild types and mutants; cases when only one of the cell types has random fitness values while the other has deterministic fitness; and cases where the mutants are advantaged or disadvantaged. Using analytical calculations and stochastic numerical simulations, we find that randomness has a strong impact on fixation time. In the case of complete graphs, randomness accelerates mutant fixation for all population sizes, and in the case of circular graphs, randomness delays mutant fixation for N larger than a threshold value (for small values of N, different behaviors are observed depending on the fitness distribution functions). These results emphasize fundamental differences in population dynamics under different assumptions on cell connectedness. They are explained by the existence of randomly occurring “dead zones” that can significantly delay fixation on networks with low connectivity; and by the existence of randomly occurring “lucky zones” that can facilitate fixation on networks of high connectivity. Results for death-birth and birth-death formulations of the Moran process, as well as for the (haploid) Wright Fisher model are presented. [ABSTRACT FROM AUTHOR]

The genetics and epigenetic processes associated with morphological organization are a principal aim of biology, ranging from cohesion between cells to shape and size of organisms. We investigate the post-embryonic development of Hypopharyngeal sclerite and cornua HPC and mandibles M of Drosophila gaucha larva. Integrated functioning of these Cephalopharyngeal skeleton parts of D. gaucha larva is essential for food acquisition, participating in locomotion and microhabitat selection. We examined two isolates by recording the growth of the HPC and M every 24 h for 8 days in parental, F1, F2 and backcross larvae. In F1 larvae, the HPC and M growth was similar to the parental. In F2 and backcross larvae, the growth was slower. Epistasis and dominance are the principal sources upon which the growth of HPC and M are based. Pleiotropic genes seem also to be involved in integrating the development of M and HPC. Our data suggest that hybridization of the isolates modified epigenetic processes involved in the development of those morphological structures of D. gaucha larva. [ABSTRACT FROM AUTHOR]

The hippocampus is the main locus of episodic memory formation and the neurons there encode the spatial map of the environment. Hippocampal place cells represent location, but their role in the learning of preferential location remains unclear. The hippocampus may encode locations independently from the stimuli and events that are associated with these locations. We have discovered a unique population code for the experience-dependent value of the context. The degree of reward-driven navigation preference highly correlates with the spatial distribution of the place fields recorded in the CA1 region of the hippocampus. We show place field clustering towards rewarded locations. Optogenetic manipulation of the ventral tegmental area demonstrates that the experience-dependent place field assembly distribution is directed by tegmental dopaminergic activity. The ability of the place cells to remap parallels the acquisition of reward context. Our findings present key evidence that the hippocampal neurons are not merely mapping the static environment but also store the concurrent context reward value, enabling episodic memory for past experience to support future adaptive behavior. [ABSTRACT FROM AUTHOR]

The integration of experimental data into genome-scale metabolic models can greatly improve flux predictions. This is achieved by restricting predictions to a more realistic context-specific domain, like a particular cell or tissue type. Several computational approaches to integrate data have been proposed—generally obtaining context-specific (sub)models or flux distributions. However, these approaches may lead to a multitude of equally valid but potentially different models or flux distributions, due to possible alternative optima in the underlying optimization problems. Although this issue introduces ambiguity in context-specific predictions, it has not been generally recognized, especially in the case of model reconstructions. In this study, we analyze the impact of alternative optima in four state-of-the-art context-specific data integration approaches, providing both flux distributions and/or metabolic models. To this end, we present three computational methods and apply them to two particular case studies: leaf-specific predictions from the integration of gene expression data in a metabolic model of Arabidopsis thaliana, and liver-specific reconstructions derived from a human model with various experimental data sources. The application of these methods allows us to obtain the following results: (i) we sample the space of alternative flux distributions in the leaf- and the liver-specific case and quantify the ambiguity of the predictions. In addition, we show how the inclusion of ℓ1-regularization during data integration reduces the ambiguity in both cases. (ii) We generate sets of alternative leaf- and liver-specific models that are optimal to each one of the evaluated model reconstruction approaches. We demonstrate that alternative models of the same context contain a marked fraction of disparate reactions. Further, we show that a careful balance between model sparsity and metabolic functionality helps in reducing the discrepancies between alternative models. Finally, our findings indicate that alternative optima must be taken into account for rendering the context-specific metabolic model predictions less ambiguous. [ABSTRACT FROM AUTHOR]

In this study, we present a theoretical framework combining experimental characterizations and analytical calculus to capture the firing rate input-output properties of single neurons in the fluctuation-driven regime. Our framework consists of a two-step procedure to treat independently how the dendritic input translates into somatic fluctuation variables, and how the latter determine action potential firing. We use this framework to investigate the functional impact of the heterogeneity in firing responses found experimentally in young mice layer V pyramidal cells. We first design and calibrate in vitro a simplified morphological model of layer V pyramidal neurons with a dendritic tree following Rall's branching rule. Then, we propose an analytical derivation for the membrane potential fluctuations at the soma as a function of the properties of the synaptic input in dendrites. This mathematical description allows us to easily emulate various forms of synaptic input: either balanced, unbalanced, synchronized, purely proximal or purely distal synaptic activity. We find that those different forms of input activity lead to various impact on the membrane potential fluctuations properties, thus raising the possibility that individual neurons will differentially couple to specific forms of activity as a result of their different firing response. We indeed found such a heterogeneous coupling between synaptic input and firing response for all types of presynaptic activity. This heterogeneity can be explained by different levels of cellular excitability in the case of the balanced, unbalanced, synchronized and purely distal activity. A notable exception appears for proximal dendritic inputs: increasing the input level can either promote firing response in some cells, or suppress it in some other cells whatever their individual excitability. This behavior can be explained by different sensitivities to the speed of the fluctuations, which was previously associated to different levels of sodium channel inactivation and density. Because local network connectivity rather targets proximal dendrites, our results suggest that this aspect of biophysical heterogeneity might be relevant to neocortical processing by controlling how individual neurons couple to local network activity. [ABSTRACT FROM AUTHOR]

Cells use surface receptors to estimate concentrations of external ligands. Limits on the accuracy of such estimations have been well studied for pairs of ligand and receptor species. However, the environment typically contains many ligands, which can bind to the same receptors with different affinities, resulting in cross-talk. In traditional rate models, such cross-talk prevents accurate inference of concentrations of individual ligands. In contrast, here we show that knowing the precise timing sequence of stochastic binding and unbinding events allows one receptor to provide information about multiple ligands simultaneously and with a high accuracy. We show that such high-accuracy estimation of multiple concentrations can be realized with simple structural modifications of the familiar kinetic proofreading biochemical network diagram. We give two specific examples of such modifications. We argue that structural and functional features of real cellular biochemical sensory networks in immune cells, such as feedforward and feedback loops or ligand antagonism, sometimes can be understood as solutions to the accurate multi-ligand estimation problem. [ABSTRACT FROM AUTHOR]

Recycling endosomes are generally thought to play a central role in endocytic recycling, but recent evidence has indicated that they also participate in other cellular events, including cytokinesis, autophagy, and neurite outgrowth. Rab small GTPases are key regulators in membrane trafficking, and although several Rab isoforms, e.g., Rab11, have been shown to regulate recycling endosomal trafficking, the precise mechanism by which these Rabs regulate recycling endosomes is not fully understood. In this study, we focused on a Rab-GTPase-activating protein (Rab-GAP), one of the key regulators of Rabs, and comprehensively screened 43 mammalian Tre-2/Bub2/Cdc16 (TBC)/Rab-GAP-domain-containing proteins (TBC proteins) for proteins that specifically localize on recycling endosomes in mouse embryonic fibroblasts (MEFs). Four of the 43 mammalian TBC proteins screened, i.e., TBC1D11, TBC1D12, TBC1D14, and EVI5, were found to colocalize well with transferrin receptor, a well-known recycling endosome marker. We further investigated the biochemical properties of TBC1D12, a previously uncharacterized TBC protein. The results showed that TBC1D12 interacted with active Rab11 through its middle region and that it did not display Rab11-GAP activity in vitro. The recycling endosomal localization of TBC1D12 was found to depend on the expression of Rab11. We also found that TBC1D12 expression had no effect on common Rab11-dependent cellular events, e.g., transferrin recycling, in MEFs and that it promoted neurite outgrowth, a specialized Rab11-dependent cellular event, of PC12 cells independently of its GAP activity. These findings indicated that TBC1D12 is a novel Rab11-binding protein that modulates neurite outgrowth of PC12 cells. [ABSTRACT FROM AUTHOR]

Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In Arabidopsis, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65 Ǻ and 2.75 Ǻ respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our in vitro biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs). [ABSTRACT FROM AUTHOR]

Creating and running realistic models of neural networks has hitherto been a task for computing professionals rather than experimental neuroscientists. This is mainly because such networks usually engage substantial computational resources, the handling of which requires specific programing skills. Here we put forward a newly developed simulation environment ARACHNE: it enables an investigator to build and explore cellular networks of arbitrary biophysical and architectural complexity using the logic of NEURON and a simple interface on a local computer or a mobile device. The interface can control, through the internet, an optimized computational kernel installed on a remote computer cluster. ARACHNE can combine neuronal (wired) and astroglial (extracellular volume-transmission driven) network types and adopt realistic cell models from the NEURON library. The program and documentation (current version) are available at GitHub repository under the MIT License (MIT). [ABSTRACT FROM AUTHOR]

Quantitative analysis of the vascular network anatomy is critical for the understanding of the vasculature structure and function. In this study, we have combined microcomputed tomography (microCT) and computational analysis to provide quantitative three-dimensional geometrical and topological characterization of the normal kidney vasculature, and to investigate how 2 core genes of the Wnt/planar cell polarity, Frizzled4 and Frizzled6, affect vascular network morphogenesis. Experiments were performed on frizzled4 (Fzd4-/-) and frizzled6 (Fzd6-/-) deleted mice and littermate controls (WT) perfused with a contrast medium after euthanasia and exsanguination. The kidneys were scanned with a high-resolution (16 μm) microCT imaging system, followed by 3D reconstruction of the arterial vasculature. Computational treatment includes decomposition of 3D networks based on Diameter-Defined Strahler Order (DDSO). We have calculated quantitative (i) Global scale parameters, such as the volume of the vasculature and its fractal dimension (ii) Structural parameters depending on the DDSO hierarchical levels such as hierarchical ordering, diameter, length and branching angles of the vessel segments, and (iii) Functional parameters such as estimated resistance to blood flow alongside the vascular tree and average density of terminal arterioles. In normal kidneys, fractal dimension was 2.07±0.11 (n = 7), and was significantly lower in Fzd4-/- (1.71±0.04; n = 4), and Fzd6-/- (1.54±0.09; n = 3) kidneys. The DDSO number was 5 in WT and Fzd4-/-, and only 4 in Fzd6-/-. Scaling characteristics such as diameter and length of vessel segments were altered in mutants, whereas bifurcation angles were not different from WT. Fzd4 and Fzd6 deletion increased vessel resistance, calculated using the Hagen-Poiseuille equation, for each DDSO, and decreased the density and the homogeneity of the distal vessel segments. Our results show that our methodology is suitable for 3D quantitative characterization of vascular networks, and that Fzd4 and Fzd6 genes have a deep patterning effect on arterial vessel morphogenesis that may determine its functional efficiency. [ABSTRACT FROM AUTHOR]

Saliva is an essential part of activities such as speaking, masticating and swallowing. Enzymes in salivary fluid protect teeth and gums from infectious diseases, and also initiate the digestion process. Intracellular calcium (Ca2+) plays a critical role in saliva secretion and regulation. Experimental measurements of Ca2+ and inositol trisphosphate (IP3) concentrations in HSY cells, a human salivary duct cell line, show that when the cells are stimulated with adenosine triphosphate (ATP) or carbachol (CCh), they exhibit coupled oscillations with Ca2+ spike peaks preceding IP3 spike peaks. Based on these data, we construct a mathematical model of coupled Ca2+ and IP3 oscillations in HSY cells and perform model simulations of three different experimental settings to forecast Ca2+ responses. The model predicts that when Ca2+ influx from the extracellular space is removed, oscillations gradually slow down until they stop. The model simulation of applying a pulse of IP3 predicts that photolysis of caged IP3 causes a transient increase in the frequency of the Ca2+ oscillations. Lastly, when Ca2+-dependent activation of PLC is inhibited, we see an increase in the oscillation frequency and a decrease in the amplitude. These model predictions are confirmed by experimental data. We conclude that, although concentrations of Ca2+ and IP3 oscillate, Ca2+ oscillations in HSY cells are the result of modulation of the IP3 receptor by intracellular Ca2+, and that the period is modulated by the accompanying IP3 oscillations. [ABSTRACT FROM AUTHOR]