Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest

Genome engineering of isogenic human ES cells to model autism disorders.

Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders, some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here, we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest

A Markovian Entropy Measure for the Analysis of Calcium Activity Time Series

Methods to analyze the dynamics of calcium activity often rely on visually distinguishable features in time series data such as spikes, waves, or oscillations. However, systems such as the developing nervous system display a complex, irregular type of calcium activity which makes the use of such methods less appropriate. Instead, for such systems there exists a class of methods (including information theoretic, power spectral, and fractal analysis approaches) which use more fundamental properties of the time series to analyze the observed calcium dynamics. We present a new analysis method in this class, the Markovian Entropy measure, which is an easily implementable calcium time series analysis method which represents the observed calcium activity as a realization of a Markov Process and describes its dynamics in terms of the level of predictability underlying the transitions between the states of the process. We applied our and other commonly used calcium analysis methods on a dataset from Xenopus laevis neural progenitors which displays irregular calcium activity and a dataset from murine synaptic neurons which displays activity time series that are well-described by visually-distinguishable features. We find that the Markovian Entropy measure is able to distinguish between biologically distinct populations in both datasets, and that it can separate biologically distinct populations to a greater extent than other methods in the dataset exhibiting irregular calcium activity. These results support the benefit of using the Markovian Entropy measure to analyze calcium dynamics, particularly for studies using time series data which do not exhibit easily distinguishable features

The genetic insulator RiboJ increases expression of insulated genes

A primary objective of synthetic biology is the construction of genetic circuits with behaviors that can be predicted based on the properties of the constituent genetic parts from which they are built. However a significant issue in the construction of synthetic genetic circuits is a phenomenon known as context dependence in which the behavior of a given part changes depending on the choice of adjacent or nearby parts. Interactions between parts compromise the modularity of the circuit, impeding the implementation of predictable genetic constructs. To address this issue, investigators have devised genetic insulators that prevent these unintended context-dependent interactions between neighboring parts. One of the most commonly used insulators in bacterial systems is the self-cleaving ribozyme RiboJ. Despite its utility as an insulator, there has been no systematic quantitative assessment of the effect of RiboJ on the expression level of downstream genetic parts. Here, we characterized the impact of insulation with RiboJ on expression of a reporter gene driven by a promoter from a library of 24 frequently employed constitutive promoters in an Escherichia coli model system. We show that, depending on the strength of the promoter, insulation with RiboJ increased protein abundance between twofold and tenfold and increased transcript abundance by an average of twofold. This result demonstrates that genetic insulators in E. coli can impact the expression of downstream genes, information that is essential for the design of predictable genetic circuits and constructs

The genetic insulator RiboJ increases expression of insulated genes

A primary objective of synthetic biology is the construction of genetic circuits with behaviors that can be predicted based on the properties of the constituent genetic parts from which they are built. However a significant issue in the construction of synthetic genetic circuits is a phenomenon known as context dependence in which the behavior of a given part changes depending on the choice of adjacent or nearby parts. Interactions between parts compromise the modularity of the circuit, impeding the implementation of predictable genetic constructs. To address this issue, investigators have devised genetic insulators that prevent these unintended context-dependent interactions between neighboring parts. One of the most commonly used insulators in bacterial systems is the self-cleaving ribozyme RiboJ. Despite its utility as an insulator, there has been no systematic quantitative assessment of the effect of RiboJ on the expression level of downstream genetic parts. Here, we characterized the impact of insulation with RiboJ on expression of a reporter gene driven by a promoter from a library of 24 frequently employed constitutive promoters in an Escherichia coli model system. We show that, depending on the strength of the promoter, insulation with RiboJ increased protein abundance between twofold and tenfold and increased transcript abundance by an average of twofold. This result demonstrates that genetic insulators in E. coli can impact the expression of downstream genes, information that is essential for the design of predictable genetic circuits and constructs

Ancient Lowland Maya neighborhoods: Average Nearest Neighbor analysis and kernel density models, environments, and urban scale

Many humans live in large, complex political centers, composed of multi-scalar communities including neighborhoods and districts. Both today and in the past, neighborhoods form a fundamental part of cities and are defined by their spatial, architectural, and material elements. Neighborhoods existed in ancient centers of various scales, and multiple methods have been employed to identify ancient neighborhoods in archaeological contexts. However, the use of different methods for neighborhood identification within the same spatiotemporal setting results in challenges for comparisons within and between ancient societies. Here, we focus on using a single method—combining Average Nearest Neighbor (ANN) and Kernel Density (KD) analyses of household groups—to identify potential neighborhoods based on clusters of households at 23 ancient centers across the Maya Lowlands. While a one-size-fits all model does not work for neighborhood identification everywhere, the ANN/KD method provides quantifiable data on the clustering of ancient households, which can be linked to environmental zones and urban scale. We found that centers in river valleys exhibited greater household clustering compared to centers in upland and escarpment environments. Settlement patterns on flat plains were more dispersed, with little discrete spatial clustering of households. Furthermore, we categorized the ancient Maya centers into discrete urban scales, finding that larger centers had greater variation in household spacing compared to medium-sized and smaller centers. Many larger political centers possess heterogeneity in household clustering between their civic-ceremonial cores, immediate hinterlands, and far peripheries. Smaller centers exhibit greater household clustering compared to larger ones. This paper quantitatively assesses household clustering among nearly two dozen centers across the Maya Lowlands, linking environment and urban scale to settlement patterns. The findings are applicable to ancient societies and modern cities alike; understanding how humans form multi-scalar social groupings, such as neighborhoods, is fundamental to human experience and social organization

Assembly of N-hexadecyl-pyridinium-4-boronic acid hexafluorophosphate monolayer films with catechol sensing selectivity

The highly water insoluble N-hexadecyl-pyridinium-4-boronic acid hexafluorophosphate is synthesised and investigated for sensor applications. This amphiphilic molecule is immobilised by evaporation of an acetonitrile solution at a basal plane pyrolytic graphite (HOPG) electrode surface and is shown to provide a monolayer film. By varying the amount of deposit partial or full coverage can be achieved. The N-hexadecyl-pyridinium-4-boronic acid hexafluorophosphate monolayer acts as an active receptor for 1,2-dihydroxy-benzene (catechol) derivatives in aqueous media. The ability to bind alizarin red S is investigated and the Langmuirian binding constant determined as a function of pH. It is shown that the immobilised boronic acid monolayer acts as sensor film for a wider range of catechols. A comparison of Langmuirian binding constants for alizarin red S (1.4 x 10(5) mol(-1) dm(3)), catechol (8.4 x 10(4) mol(-1) dm(3)), caffeic acid (7.5 x 10(4) mol(-1) dm(3)), dopamine (1.0 x 10(4) mol(-1) dm(3)), and L-dopa (8 x 10(3) mol(-1) dm(3)) reveals that a combination of hydrophobicity and electrostatic interaction causes considerable selectivity effects.China Scholarship Counci