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.

Accumulating evidence over the past three decades suggests that altered calcium signaling during development may be a major driving force for adult pathophysiological events. Well over a hundred human genes encode proteins that are specifically dedicated to calcium homeostasis and calcium signaling, and the majority of these are expressed during embryonic development. Recent advances in molecular techniques have identified impaired calcium signaling during development due to either mutations or dysregulation of these proteins. This impaired signaling has been implicated in various human diseases ranging from cardiac malformations to epilepsy. Although the molecular basis of these and other diseases have been well studied in adult systems, the potential developmental origins of such diseases are less well characterized. In this review, we will discuss the recent evidence that examines different patterns of calcium activity during early development, as well as potential medical conditions associated with its dysregulation. Studies performed using various model organisms, including zebrafish, Xenopus, and mouse, have underscored the critical role of calcium activity in infertility, abortive pregnancy, developmental defects, and a range of diseases which manifest later in life. Understanding the underlying mechanisms by which calcium regulates these diverse developmental processes remains a challenge; however, this knowledge will potentially enable calcium signaling to be used as a therapeutic target in regenerative and personalized medicine.

The intracellular and intercellular flux of calcium ions represents an ancient and universal mode of signaling that regulates an extensive array of cellular processes. Evidence for the central role of calcium signaling includes various techniques that allow the visualization of calcium activity in living cells. While extensively investigated in mature cells, calcium activity is equally important in developing cells, particularly the embryonic nervous system where it has been implicated in a wide variety array of determinative events. However, unlike in mature cells, where the calcium dynamics display regular, predictable patterns, calcium activity in developing systems is far more sporadic, irregular, and diverse. This renders the ability to assess calcium activity in a consistent manner extremely challenging, challenges reflected in the diversity of methods employed to analyze calcium activity in neural development. Here we review the wide array of calcium detection and analysis methods used across studies, limiting the extent to which they can be comparatively analyzed. The goal is to provide investigators not only with an overview of calcium activity analysis techniques currently available, but also to offer suggestions for future work and standardization to enable informative comparative evaluations of this fundamental and important process in neural development.

Calcium is a ubiquitous signaling molecule that plays a vital role in many physiological processes. Recent work has shown that calcium activity is especially critical in vertebrate neural development. Here, we investigated if calcium activity and neuronal phenotype are correlated only on a population level or on the level of single cells. Using Xenopus primary cell culture in which individual cells can be unambiguously identified and associated with a molecular phenotype, we correlated calcium activity with neuronal phenotype on the single-cell level. This analysis revealed that, at the neural plate stage, a high frequency of low-amplitude spiking activity correlates with an excitatory, glutamatergic phenotype, while high-amplitude spiking activity correlates with an inhibitory, GABAergic phenotype. Surprisingly, we also found that high-frequency, low-amplitude spiking activity correlates with neural progenitor cells and that differentiating cells exhibit higher spike amplitude. Additional methods of analysis suggested that differentiating marker tubb2b-expressing cells exhibit relatively persistent and predictable calcium activity compared to the irregular activity of neural progenitor cells. Our study highlights the value of using a range of thresholds for analyzing calcium activity data and underscores the importance of employing multiple methods to characterize the often irregular, complex patterns of calcium activity during early neural development.

Phage satellites are mobile genetic elements that parasitize a helper phage to complete their life cycle. While phage satellites are widespread in diverse bacterial hosts, none have yet been isolated in Mycobacteriaceae. Here, we report the first phage satellites isolated and characterized in Mycobacteriaceae, Extracellular Prophage-Inducing Particles (EPIPs). EPIPs induce a helper phage-HerbertWM, a Mycolicibacterium aichiense prophage-upon infection. Genomic sequencing of thirteen isolates revealed a genome size of ∼11 kb and ∼83.3% nucleotide similarity calculated using Virus Intergenomic Distance Calculator (VIRIDIC). Although gene content and synteny were broadly conserved across the isolates, the EPIPs were categorized into distinct groups based on VIRIDIC similarity scores, gene content, and synteny. Notably, EPIPs lack capsid proteins, tail proteins, and holins, suggesting EPIPs hijack the machinery of HerbertWM to replicate, assemble, and lyse the host. Furthermore, the unique gene content of EPIPs compared to other phage satellites, particularly the presence of a tape measure protein and absence of antirepressors, suggests they are distinct from other phage satellite classes. Transmission electron microscopy of EPIPs reveals that they not only feature smaller capsids compared to HerbertWM, but that they feature highly flexible tails of variable length. Additionally, greater EPIP protein similarity to proteins in bacteria as opposed to free phage proteins suggests a functional or evolutionary relationship may exist. The nucleotide similarity, synteny, and unique gene content of EPIPs suggest EPIPs are a distinct class of phage satellites that induce Mycobacteriaceae prophages.