Population genomic response to geographic gradients by widespread and endemic fishes of the Arabian Peninsula

Genetic structure within marine species may be driven by local adaptation to their environment, or alternatively by historical processes, such as geographic isolation. The gulfs and seas bordering the Arabian Peninsula offer an ideal setting to examine connectivity patterns in coral reef fishes with respect to environmental gradients and vicariance. The Red Sea is characterized by a unique marine fauna, historical periods of desiccation and isolation, as well as environmental gradients in salinity, temperature, and primary productivity that vary both by latitude and by season. The adjacent Arabian Sea is characterized by a sharper environmental gradient, ranging from extensive coral cover and warm temperatures in the southwest, to sparse coral cover, cooler temperatures, and seasonal upwelling in the northeast. Reef fish, however, are not confined to these seas, with some Red Sea fishes extending varying distances into the northern Arabian Sea, while their pelagic larvae are presumably capable of much greater dispersal. These species must therefore cope with a diversity of conditions that invoke the possibility of steep clines in natural selection. Here, we test for genetic structure in two widespread reef fish species (a butterflyfish and surgeonfish) and eight range‐restricted butterflyfishes across the Red Sea and Arabian Sea using genome‐wide single nucleotide polymorphisms. We performed multiple matrix regression with randomization analyses on genetic distances for all species, as well as reconstructed scenarios for population subdivision in the species with signatures of isolation. We found that (a) widespread species displayed more genetic subdivision than regional endemics and (b) this genetic structure was not correlated with contemporary environmental parameters but instead may reflect historical events. We propose that the endemic species may be adapted to a diversity of local conditions, but the widespread species are instead subject to ecological filtering where different combinations of genotypes persist under divergent ecological regimes.     

Comparative biokinetics of trivalent radionuclides with similar ionic dimensions: promethium-147, curium-242 and americium-241

Data on the distribution and redistribution patterns in the laboratory rat of three trivalent elements with a similar ionic radius have been compared. This showed that these distributions for the two ions with the same ionic radius (111 pm), i.e., those of promethium (a lanthanoid) and curium (an actinoid), were indistinguishable and that americium, with a slightly larger ion size (111.5 pm), behaved similarly. The results are consistent with the suggestion that ion size is the only important factor controlling the deposition and redistribution patterns of trivalent lanthanoids and actinoids in rats. The result is important because it suggests that the same radiological protection dosimetry models should be used for trivalent actinoids and lanthanoids, that human volunteer data generated for lanthanoid isotopes can be used to predict the behavior of actinoids with the same ion size, and that appropriate pairs of beta-particle-emitting lanthanoid and alpha-particle-emitting actinoids could be used to study the relative toxicity of alpha and beta particles in experimental animals.     

Intercellular coupling confers robustness against mutations in the SCN circadian clock network

Molecular mechanisms of the mammalian circadian clock have been studied primarily by genetic perturbation and behavioral analysis. Here, we used bioluminescence imaging to monitor Per2 gene expression in tissues and cells from clock mutant mice. We discovered that Per1 and Cry1 are required for sustained rhythms in peripheral tissues and cells, and in neurons dissociated from the suprachiasmatic nuclei (SCN). Per2 is also required for sustained rhythms, whereas Cry2 and Per3 deficiencies cause only period length defects. However, oscillator network interactions in the SCN can compensate for Per1 or Cry1 deficiency, preserving sustained rhythmicity in mutant SCN slices and behavior. Thus, behavior does not necessarily reflect cell-autonomous clock phenotypes. Our studies reveal previously unappreciated requirements for Per1, Per2, and Cry1 in sustaining cellular circadian rhythmicity and demonstrate that SCN intercellular coupling is essential not only to synchronize component cellular oscillators but also for robustness against genetic perturbations.     

Real-Time Imaging of Electrical Signals with an Infrared FDA-Approved Dye

Clinical methods used to assess the electrical activity of excitable cells are often limited by their poor spatial resolution or their invasiveness. One promising solution to this problem is to optically measure membrane potential using a voltage-sensitive dye, but thus far, none of these dyes have been available for human use. Here we report that indocyanine green (ICG), an infrared fluorescent dye with FDA approval as an intravenously administered contrast agent, is voltage-sensitive. The fluorescence of ICG can follow action potentials in artificial neurons and cultured rat neurons and cardiomyocytes. ICG also visualized electrical activity induced in living explants of rat brain. In humans, ICG labels excitable cells and is routinely visualized transdermally with high spatial resolution. As an infrared voltage-sensitive dye with a low toxicity profile that can be readily imaged in deep tissues, ICG may have significant utility for clinical and basic research applications previously intractable for potentiometric dyes.Voltage-sensitive dyes provide a way to observe cellular electrical activity without the physical limitations imposed by electrodes. Although these dyes can monitor membrane potential with a resolution of a few microns from large populations of cells (1), there are three obstacles that prevent the use of these dyes in many research settings, including clinical research:

• 1.Most voltage-sensitive dyes use visible wavelengths of light that prevent imaging of tissues beneath the skin.
• 2.Many of these dyes produce significant toxicity or off-target effects (2).
• 3.Before this report, to our knowledge, no voltage-sensitive dyes have ever been available for administration in humans, which has limited their value in biomedically focused research.

Here, we show that indocyanine green (ICG), an FDA-approved fluorescent dye routinely used in many clinical tests, is voltage-sensitive. Our initial experimental system used Xenopus laevis oocytes. Changes in the membrane potential of the cell induced by two-electrode voltage-clamp resulted in robust, consistent changes in the fluorescence of ICG (Fig. 1, inset). All data in this work was obtained from single acquisitions with no averaging of multiple images. The voltage-dependent fluorescence changes were roughly linear with respect to membrane potential and had a magnitude of ∼1.9% of the baseline fluorescence per 100 mV of membrane potential change (Fig. 1). Additionally, ICG displayed a rapid response with a primary time constant of 4 ms (see Fig. S1 in the Supporting Material), suggesting that this dye could successfully monitor action potentials.Open in a separate windowFigure 1ICG-labeled oocytes showed that ICG’s fluorescence (blue points) is roughly linearly dependent (red line, fit to data) with voltage. (Inset) Oocyte membrane potential was held at −60 mV and then pulsed to potentials ranging from −120 mV (blue) to +120 mV (red). Ex: 780 nm, Em: 818–873 nm.To test this hypothesis, we transformed our oocytes into synthetic neurons, previously dubbed “excitocytes”, by coinjecting them with cRNA of voltage-gated sodium (Na_v) and potassium channel components (3). Under suitable current-clamp conditions, excitocytes fire trains of action potentials similar to those in naturally excitable cells. ICG’s fluorescence clearly recapitulated action potentials firing at speeds above 100 Hz (Fig. 2 A), faster than the physiological firing rates of most neurons (4).Open in a separate windowFigure 2ICG can monitor action potentials. (A) Oocytes coinjected with voltage-gated sodium and potassium channel cRNA fired action potentials (bottom, green) when held under current clamp. ICG fluorescence changes (top, blue) detected these action potentials at a rate of 107 Hz. Stimulus start (black arrow) and end (red arrow) are shown. (B and C) ICG fluorescence (blue, inverted) distinguished between healthy action potentials from wild-type sodium channels (B, green) and diseased action potentials from sodium channels with a myotonic substitution (C, green). Cells are stimulated for the entire time course of these panels. The delay between action potentials and the ICG signal is due to a low-pass filtering effect caused by the dye response time and the camera integration time. (D) In cells with myotonic sodium channels, a brief stimulus (top, black) was sufficient to elicit a train of action potentials (bottom, green) that only ceased upon significant hyperpolarization, as expected in a myotonia. ICG fluorescence (middle, blue) successfully followed each one of these action potentials.We extended the excitocyte technique from wild-type channels to evaluation of channelopathies and their effects on excitability to determine whether ICG could discriminate between normal and diseased action potentials based on shape. We compared excitocytes injected with wild-type Na_v channel cRNA to those injected with cRNA coding for a version of Na_v channel containing a point mutation, G1306E, which produces episodic myotonia (5). This disease is characterized by continued action potential firing in skeletal muscles after cessation of voluntary stimuli; the resulting prolonged muscle contractions are the hallmark of myotonia. Compared to the wild-type Na_v channel, the G1306E mutation causes a slowing of the fast inactivation of the Na_v channels, which in turn results in broadened action potentials (5). The electrical recordings and the ICG fluorescence response clearly distinguished the sharp action potentials produced by the healthy sodium channel (Fig. 2 B, and see Fig. S2) from the wider peaks produced by the myotonic sodium channel (Fig. 2 C, and see Fig. S2). Furthermore, a brief injection of current led to repetitive firing and hyperexcitability that persisted after the stimulus was stopped. ICG fluorescence clearly resolved every action potential of this myotonia-like behavior (Fig. 2 D). The successful recreation of disease-like action potentials validates the excitocyte system as a convenient method for investigating the electrophysiological effects of channelopathies.We next investigated whether ICG’s voltage sensitivity extended to excitable mammalian tissue. This validation was critical, inasmuch as other voltage-sensitive dyes have shown promise in invertebrate preparations but had much smaller signals in mammalian cells (6). We first measured ICG fluorescence from cultured rat dorsal root ganglion neurons. Under whole-cell current clamp, we observed neurons firing in the stereotypical fashion of the nociceptive C-type fiber, and these action potentials were clearly visible in the ICG fluorescence (Fig. 3 A, and see Fig. S3). We also examined syncytia of cultured cardiomyocytes from neonatal rats (7) to further validate ICG’s utility; these cells beat spontaneously and showed changes in ICG fluorescence indicative of changes in membrane potential (Fig. 3 B). Although we cannot formally exclude the possibility that the cardiomyocytes’ physical motion produced fluorescence changes, several observations suggested that these effects were minimal (see Fig. S4). Taken together, our results in frog and rat cells confirmed that ICG voltage sensitivity was broadly applicable across a range of tissues and not confined to a particular animal or cell lineage.Open in a separate windowFigure 3ICG follows electrical activity in living mammalian tissue. (A) Rat cultured dorsal root ganglion cells under current-clamp (black arrow, pulse start; red arrow, pulse end) fired action potentials (green), that ICG fluorescence tracked (blue, inverted, low-pass-filtered at 225 Hz; blue arrow, relative fluorescence change). (B) ICG fluorescence sensed spontaneous membrane potential changes in cardiomyocyte syncytia. (C) In rat brain slices, ICG responds differently to no stimulus (black) and stimuli of increasing intensity (magenta, cyan, green, and blue, increasing amplitude; scale bar shows relative fluorescence change). Weaker stimuli traces (e.g., magenta) show complete fluorescence recovery whereas larger stimuli (e.g., blue) do not fully recover within this time course; traces are vertically offset for clarity. (D) Tetrodotoxin (TTX) reduced the ICG response to a stimulus over 12 min (green, pre-TTX; cyan, magenta, and black, increasing time post-TTX; low-pass-filtered at 40 Hz; black arrow, stimulus).Finally, we tested whether ICG voltage sensitivity could be detected in a complex tissue. Rat hippocampal slice cultures comprise a well-described organotypic preparation in which the three-dimensional architecture, neuronal connections, and glial interactions are maintained (8,9). Using these rat brain explants, we found that brain excitation produced by field electrode stimulation was clearly accompanied by ICG fluorescence changes (see Fig. S5). Additionally, ICG discriminated between electrical responses caused by differing excitation intensities and durations (Fig. 3 C, and see Fig. S5). To confirm that the fluorescence changes originated from changes in excitable cell activity, we used the Na_v channel blocker tetrodotoxin (TTX). When applied to brain slices, electrical excitability was clearly inhibited (Fig. 3 D, and see Fig. S5) and partial recovery was observed upon subsequent TTX removal (see Fig. S5). These signals measuring brain slice activity were similar in shape and magnitude to those reported using other voltage-sensitive dyes (10,11). This demonstrates that ICG can report on electrical activity even in a physiological architecture with many nonexcitable cells.To our knowledge, this is the first report that a clinically approved fluorescent dye is voltage-sensitive. Our results demonstrate that indocyanine green can accurately detect action potentials at firing rates common in mammalian neurons, and that it is sensitive enough to distinguish between healthy and diseased action potentials in a model system. ICG can measure electrical activity in mammalian neurons, cardiomyocytes, and explanted brain tissue. This voltage sensitivity was observed with both monochromatic and broad-band illumination sources (data not shown), under labeling conditions that differed in solution composition, duration, and dye concentration (see Methods in the Supporting Material), and at temperatures ranging from 19°C to 30°C. ICG’s water solubility further extends its potential utility. This robustness suggests that ICG can be used to measure voltage in many environments and tissues.ICG has been FDA-approved for use in ophthalmic angiography, as well as in tests of cardiac output and hepatic function (12) and is additionally used off-label in a number of surgical applications (13). Interestingly, ICG has been shown to clearly label retinal ganglion cells in human patients (see Fig. S6) (14). This provides immediate motivation for biomedical investigations, because laboratory findings with ICG can potentially be translated to humans. Although many other voltage-sensitive dyes have been described, some with similar structures to ICG (15) and others with faster or larger signals (15,16), as of this writing none of these are FDA-approved. Additionally, ICG utilizes wavelengths further into the infrared spectrum than other available fast potentiometric dyes (17) and can thus be imaged in tissues up to 2 cm deep (18). This presents the possibility of optically imaging electrical activity deeper inside tissues than is feasible today. Although two-photon excitation with voltage-sensitive dyes can improve imaging depth, it remains intrinsically limited by the unaffected emission wavelength (19). Finally, ICG has been used in patients for more than 50 years and is known to have low toxicity (18,20). These properties suggest that ICG voltage sensitivity could extend the capabilities of modern electrophysiological techniques for disease diagnosis and monitoring in the clinic, and allow for the investigation of previously inaccessible experimental systems in basic research.     

Real-Time Imaging of Electrical Signals with an Infrared FDA-Approved Dye

Clinical methods used to assess the electrical activity of excitable cells are often limited by their poor spatial resolution or their invasiveness. One promising solution to this problem is to optically measure membrane potential using a voltage-sensitive dye, but thus far, none of these dyes have been available for human use. Here we report that indocyanine green (ICG), an infrared fluorescent dye with FDA approval as an intravenously administered contrast agent, is voltage-sensitive. The fluorescence of ICG can follow action potentials in artificial neurons and cultured rat neurons and cardiomyocytes. ICG also visualized electrical activity induced in living explants of rat brain. In humans, ICG labels excitable cells and is routinely visualized transdermally with high spatial resolution. As an infrared voltage-sensitive dye with a low toxicity profile that can be readily imaged in deep tissues, ICG may have significant utility for clinical and basic research applications previously intractable for potentiometric dyes.     

HORMESIS RESULTS IN TRADE‐OFFS WITH IMMUNITY

Many have argued that we may be able to extend life and improve human health through hormesis, the beneficial effects of low‐level toxins and other stressors. But, studies of hormesis in model systems have not yet established whether stress‐induced benefits are cost free, artifacts of inbreeding, or come with deleterious side effects. Here, we provide evidence that hormesis results in trade‐offs with immunity. We find that a single topical dose of dead spores of the entomopathogenic fungus, Metarhizium robertsii, increases the longevity of the fruit fly, Drosophila melanogaster, without significant decreases in fecundity. We find that hormetic benefits of pathogen challenge are greater in lines that lack key components of antifungal immunity (Dif and Turandot M). And, in outbred fly lines, we find that topical pathogen challenge enhances both survival and fecundity, but reduces ability to fight off live infections. The results provide evidence that hormesis is manifested by stress‐induced trade‐offs with immunity, not cost‐free benefits or artifacts of inbreeding. Our findings illuminate mechanisms underlying pathogen‐induced life‐history trade‐offs, and indicate that reduced immune function may be an ironic side effect of the “elixirs of life.”     

tigaR: integrative significance analysis of temporal differential gene expression induced by genomic abnormalities

Background

To determine which changes in the host cell genome are crucial for cervical carcinogenesis, a longitudinal in vitro model system of HPV-transformed keratinocytes was profiled in a genome-wide manner. Four cell lines affected with either HPV16 or HPV18 were assayed at 8 sequential time points for gene expression (mRNA) and gene copy number (DNA) using high-resolution microarrays. Available methods for temporal differential expression analysis are not designed for integrative genomic studies.

Results

Here, we present a method that allows for the identification of differential gene expression associated with DNA copy number changes over time. The temporal variation in gene expression is described by a generalized linear mixed model employing low-rank thin-plate splines. Model parameters are estimated with an empirical Bayes procedure, which exploits integrated nested Laplace approximation for fast computation. Iteratively, posteriors of hyperparameters and model parameters are estimated. The empirical Bayes procedure shrinks multiple dispersion-related parameters. Shrinkage leads to more stable estimates of the model parameters, better control of false positives and improvement of reproducibility. In addition, to make estimates of the DNA copy number more stable, model parameters are also estimated in a multivariate way using triplets of features, imposing a spatial prior for the copy number effect.

Conclusion

With the proposed method for analysis of time-course multilevel molecular data, more profound insight may be gained through the identification of temporal differential expression induced by DNA copy number abnormalities. In particular, in the analysis of an integrative oncogenomics study with a time-course set-up our method finds genes previously reported to be involved in cervical carcinogenesis. Furthermore, the proposed method yields improvements in sensitivity, specificity and reproducibility compared to existing methods. Finally, the proposed method is able to handle count (RNAseq) data from time course experiments as is shown on a real data set.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-327) contains supplementary material, which is available to authorized users.     

CT694 and pgp3 as Serological Tools for Monitoring Trachoma Programs

Background

Defining endpoints for trachoma programs can be a challenge as clinical signs of infection may persist in the absence of detectable bacteria. Antibody-based tests may provide an alternative testing strategy for surveillance during terminal phases of the program. Antibody-based assays, in particular ELISAs, have been shown to be useful to document C. trachomatis genital infections, but have not been explored extensively for ocular C. trachomatis infections.

Methodology/Principal Findings

An antibody-based multiplex assay was used to test two C. trachomatis antigens, pgp3 and CT694, for detection of trachoma antibodies in bloodspots from Tanzanian children (n = 160) collected after multiple rounds of mass azithromycin treatment. Using samples from C. trachomatis-positive (by PCR) children from Tanzania (n = 11) and control sera from a non-endemic group of U.S. children (n = 122), IgG responses to both pgp3 and CT694 were determined to be 91% sensitive and 98% specific. Antibody responses of Tanzanian children were analyzed with regard to clinical trachoma, PCR positivity, and age. In general, children with more intense ocular pathology (TF/TI = 2 or most severe) had a higher median antibody response to pgp3 (p = 0.0041) and CT694 (p = 0.0282) than those with normal exams (TF/TI = 0). However, 44% of children with no ocular pathology tested positive for antibody, suggesting prior infection. The median titer of antibody responses for children less than three years of age was significantly lower than those of older children. (p<0.0001 for both antigens).

Conclusions/Significance

The antibody-based multiplex assay is a sensitive and specific additional tool for evaluating trachoma transmission. The assay can also be expanded to include antigens representing different diseases, allowing for a robust assay for monitoring across NTD programs.