Developmental toxicity of ethanol in Drosophila melanogaster

Ethanol was tested for teratogenicity in Drosophila melanogaster. Treatment consisted of rearing the fly larvae in media containing initial ethanol concentrations of 0%, 4%, 8%, or 14% by weight. Emerging flies were inspected for gross malformations. A low frequency of malformations was seen among controls (0.82%), increasing to 10.36% of emerging adults at the highest ethanol dose. The most common malformation involved the legs (segments missing or distorted or complete absence) and wings (uninflated, distorted, or absent). Less frequent defects included fused or missing mouth parts and missing halteres. Also, by exposing staged larvae to ethanol and examining the emerging flies, developmental stage sensitivity of Drosophila was investigated in terms of timing of treatment initiation. The results suggested that the incidence of defects increased with length of exposure. These results support the assumption that ethanol itself is the causative agent in ethanol-induced developmental toxicity and further support the use of Drosophila for developmental toxicity screening.     

Optical coherence tomography as a noninvasive 3D real time imaging tool for the rapid evaluation of phenotypic variations in insect embryonic development

Noninvasive visualization of embryos at different development stages is crucial for the understanding of the basic developmental biology. It is therefore desirable to have an imaging tool capable of rapidly evaluating the effects of gene manipulation or genome editing in developing embryos for the studies of gene functions and genetic engineering. Here, we propose and demonstrate a novel use of optical coherence tomography (OCT) to noninvasively exam the embryonic development of the migratory locusts in real time with 3‐dimensional (3D) view capability. In particular, we obtain the sufficiently high spatial resolution tomographic 2D and 3D images of live locust embryos throughout their development processes. We show that not only we are able to noninvasively observe all previously known forms of blastokinesis as an embryo develops, such as anatrepsis, katatrepsis, revolution, rotation and diapauses, and determine their precise occurring time or duration, but also discover an unreported rotation form we named “twist.” In addition, with the OCT images we determined the exact occurring time of diapauses of the locusts from Tibetan plateau for the first time. Finally, we demonstrate that OCT systems can be used to rapidly capture the development defects of genetically modified embryos in which certain genes essential for embryonic development were suppressed by RNA interference. Our work shows that OCT is an enabling imaging tool with sufficient spatial resolution for the rapid evaluation of embryonic variations of small animals.     

Inheritance of OCT4 predetermines fate choice in human embryonic stem cells

It is well known that clonal cells can make different fate decisions, but it is unclear whether these decisions are determined during, or before, a cell's own lifetime. Here, we engineered an endogenous fluorescent reporter for the pluripotency factor OCT4 to study the timing of differentiation decisions in human embryonic stem cells. By tracking single‐cell OCT4 levels over multiple cell cycle generations, we found that the decision to differentiate is largely determined before the differentiation stimulus is presented and can be predicted by a cell's preexisting OCT4 signaling patterns. We further quantified how maternal OCT4 levels were transmitted to, and distributed between, daughter cells. As mother cells underwent division, newly established OCT4 levels in daughter cells rapidly became more predictive of final OCT4 expression status. These results imply that the choice between developmental cell fates can be largely predetermined at the time of cell birth through inheritance of a pluripotency factor.     

A feasibility study of OCT for anatomical and vascular phenotyping of mouse embryo

The embryo phenotyping of genetic murine model is invaluable when investigating functions of genes underlying embryonic development and birth defect. Although traditional imaging technologies such as ultrasound are very useful for evaluating phenotype of murine embryos, the use of advanced techniques for phenotyping is desirable to obtain more information from genetic research. This letter tests the feasibility of optical coherence tomography (OCT) as a high‐throughput phenotyping tool for murine embryos. Three‐dimensional OCT imaging is performed for live and cleared mouse embryos in the late developmental stage (embryonic day 17.5). By using a dynamic focusing method and OCT angiography (OCTA) approach, our OCT imaging of the embryo exhibits rapid and clean visualization of organ structures deeper than 5 mm and complex microvasculature of perfused blood vessels in the murine embryonic body. This demonstration suggests that OCT imaging can be useful for comprehensively assessing embryo anatomy and angiography of genetically engineered mice.     

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue². In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study^3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.     

Optical coherence tomography image denoising using a generative adversarial network with speckle modulation

Optical coherence tomography (OCT) is widely used for biomedical imaging and clinical diagnosis. However, speckle noise is a key factor affecting OCT image quality. Here, we developed a custom generative adversarial network (GAN) to denoise OCT images. A speckle‐modulating OCT (SM‐OCT) was built to generate low speckle images to be used as the ground truth. In total, 210 000 SM‐OCT images were used for training and validating the neural network model, which we call SM‐GAN. The performance of the SM‐GAN method was further demonstrated using online benchmark retinal images, 3D OCT images acquired from human fingers and OCT videos of a beating fruit fly heart. The denoise performance of the SM‐GAN model was compared to traditional OCT denoising methods and other state‐of‐the‐art deep learning based denoise networks. We conclude that the SM‐GAN model presented here can effectively reduce speckle noise in OCT images and videos while maintaining spatial and temporal resolutions.     

Deciphering the role of Shh signaling in axial defects produced by ethanol exposure

BACKGROUND: The phenotype of embryos exposed to ethanol is complex and likely due to multiple alterations in developmental pathways. We have previously demonstrated that Sonic hedgehog signaling (Shh‐s) was reduced in both chicken and zebrafish embryos when exposed to ethanol. METHODS: There are many tissues affected by embryonic ethanol exposure, and in this article we explore the development of axial tissues, using zebrafish embryos. We then compare these effects to the phenotypes produced by exposure to two drugs that also inhibit Shh‐s: cyclopamine and forskolin. RESULTS: We found alterations in the development of the notochord and somites produced by all three compounds, although only ethanol produced developmental delay of epiboly. Upon observation of early developing embryos, muscle pioneer cells were completely lost in cyclopamine‐treated embryos, and reduced, but less so, in embryos treated with forskolin and ethanol. Ethanol treatment produced a dose‐dependent reduction in total body length that may be linked to epiboly delay seen earlier during development. Despite the differences between cyclopamine and forskolin, we found that shh mRNA injection rescued the short body length, the alteration in somite shape, and the cyclopia produced by ethanol exposure. CONCLUSIONS: Taken together, each teratogen produced a unique set of phenotypic changes in the body axis, suggesting that each compound affects Shh‐s and also produces a distinctive set of molecular alterations. However, addition of exogenous Shh to ethanol treated zebrafish prevented many of the gross physical phenotypes, suggesting that the suppression of Shh‐s is one of the major effects of ethanol exposure. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Prenatal Stress and Development: Beyond The Single Cause and Effect Paradigm

Our awareness of the causes of stress‐induced developmental dysfunction has increased dramatically over the past decade, and it is becoming increasingly clear that a number of factors can have considerable impacts on the developing fetus. Although there is a tendency in investigations of developmental teratogens to attribute specific causes to adverse fetal outcomes, it is important we recognize that for most developmental dysfunctions it is unlikely a single cause, but yet a series of environmental insults combined with genetic predisposition that ultimately leads to a disease state. Nonetheless, a number of developmental teratogens, such as maternal psychological stress and chemical exposures, have been shown to increase the likelihood of developmental defects. These defects can manifest during development, leading to observable birth defects, or could become evident long after birth, even into adulthood. In addition, epigenetic mutations in the germline can alter the phenotype of successive generations through transgenerational inheritance, and in this way environmental factors can alter the developmental outcomes and disease predispositions of future generations. Understanding this complexity is essential to interpretations of causality in the studies of stress‐induced developmental dysfunction and needs to be fully considered to more effectively interpret potential outcomes. Birth Defects Research (Part C) 96:289–298, 2012. © 2013 Wiley Periodicals, Inc.     

Optical Coherence Tomography for live imaging of mammalian development

Understanding the nature and mechanism of congenital defects of the different organ systems in humans has heavily relied on the analysis of the corresponding mutant phenotypes in rodent models. Optical Coherence Tomography (OCT) has recently emerged as a powerful tool to study early embryonic development. This non-invasive optical methodology does not require labeling and allows visualization of embryonic tissues with single cell resolution. Here, we will discuss how OCT can be applied for structural imaging of early mouse and rat embryos in static culture, cardiodynamic and blood flow analysis, and in utero embryonic imaging at later stages of gestation, demonstrating how OCT can be used to assess structural and functional birth defects in mammalian models.     

The telomeric protein SNM1B/Apollo is required for normal cell proliferation and embryonic development

Conserved metallo β‐Lactamase and β‐CASP (CPSF‐Artemis‐Snm1‐Pso2) domain nuclease family member SNM1B/Apollo is a shelterin‐associated protein that localizes to telomeres through its interaction with TRF2. To study its in vivo role, we generated a knockout of SNM1B/Apollo in a mouse model. Snm1B/Apollo homozygous null mice die at birth with developmental delay and defects in multiple organ systems. Cell proliferation defects were observed in Snm1B/Apollo mutant mouse embryonic fibroblasts (MEFs) owing to high levels of telomeric end‐to‐end fusions. Deficiency of the nonhomologous end‐joining (NHEJ) factor Ku70, but not p53, rescued the developmental defects and lethality observed in Snm1B/Apollo mutant mice as well as the impaired proliferation of Snm1B/Apollo‐deficient MEFs. These findings demonstrate that SNM1B/Apollo is required to protect telomeres against NHEJ‐mediated repair, which results in genomic instability and the consequent multi‐organ developmental failure. Although Snm1B/Apollo‐deficient MEFs exhibited high levels of apoptosis, abrogation of p53‐dependent programmed cell death did not rescue the multi‐organ developmental failure in the mice.     

Antimelanogenic Activity of Ocotillol‐Type Saponins from Panax vietnamensis

The ocotillol (OCT)‐type saponins have been known as a tetracyclic triterpenoid, possessing five‐ or six‐membered epoxy ring in the side chain. Interestingly, this type saponin was mostly found in Panax vietnamensis Ha et Grushv ., Araliaceae (VG), hence making VG unique from the other Panax spp. Five OCT‐type saponins, majonoside R2, vina‐ginsenoside R2, majonoside R1, pseudoginsenoside RT4, vina‐ginsenoside R11, together with three protopanaxadiol (PPD)‐type saponins and four protopanaxatriol (PPT)‐type saponins from VG were evaluated for their antimelanogenic activity. All of isolates were found to be active. More importantly, the five OCT‐type saponins inhibited melanin production in B16‐F10 mouse melanoma cells, without showing any cytotoxicity. Besides ginsenoside Rd and ginsenoside Rg3 in PPD and notoginsenoside R1 in PPT‐type saponins, majonoside R2 was the most potent melanogenesis inhibitory activity in OCT‐type saponins. In this article, we highlighted antimelanogenic activity of OCT‐type saponins and potential structure–activity relationship (SAR) of ginsenosides. Our results suggested that OCT‐type saponins could be used as a depigmentation agent.     

Teratogenic interaction of ethanol and hyperthermia in mice

Alcohol and maternal hyperthermia have been implicated in human birth defects. Both ethanol and heat can induce neural tube defects (NTDs) and other developmental abnormalities in mice when large doses are given during pregnancy. To explore the teratogenic interaction of both agents, pregnant ICR mice were injected with a single dose of 25% ethanol and/or were heat-stressed in a water bath at 42 degrees C on the morning of Day 8 of gestation. Combined treatment with ethanol (0.01-0.02 ml/g) and heat (10 min), when they were given concurrently or 1 hr apart, resulted in a significant increase of resorptions and externally malformed fetuses. Skeletal malformations and visceral variations also increased significantly following a concurrent exposure to both agents. These results indicate that ethanol and heat can be synergistically teratogenic in mice when the doses of each agent are below the teratogenic threshold. It was also suggested that pretreatment with a small dose of ethanol may not enhance the teratogenicity of heat when the hyperthermic stress is strong enough and teratogenic by itself.     

Bowman's topography for improved detection of early ectasia

The aim of this study was to evaluate whether OCT topography of the Bowman's layer and artificial intelligence (AI) can result in better diagnosis of forme fruste (FFKC) and clinical keratoconus (KC). Normal (n = 221), FFKC (n = 72) and KC (n = 116) corneas were included. Some of the FFKC and KC patients had the fellow eye (VAE‐NT) with normal topography (n = 30). The Scheimpflug and OCT scans of the cornea were analyzed. The curvature and surface aberrations (ray tracing) of the anterior corneal surface [air‐epithelium (A‐E) interface in OCT] and epithelium‐Bowman's layer (E‐B) interface (in OCT only) were calculated. Four random forest models were constructed: (1) Scheimpflug only; (2) OCT A‐E only; (3) OCT E‐B only; (4) OCT A‐E and E‐B combined. For normal eyes, both Scheimpflug and OCT (A‐E and E‐B combined) performed equally in identifying these eyes (P = .23). However, OCT A‐E and E‐B showed that most VAE‐NT eyes were topographically similar to normal eyes and did not warrant a separate classification based on topography alone. For identifying FFKC eyes, OCT A‐E and E‐B combined performed significantly better than Scheimpflug (P = .006). For KC eyes, both Scheimpflug and OCT performed equally (P = 1.0). Thus, OCT Topography of Bowman's layer significantly improved the detection of FFKC eyes.     

Tissue- and developmental stage-specific expression of the rat ornithine carbamoyltransferase gene in transgenic mice

Rat ornithine carbamoyltransferase (OCT; EC 2.1.3.3) is encoded by a large gene of 75 kilobases. Expression of this gene is restricted to the liver and small intestine, and there is an increase in expression late in gestation. The recombinant gene carrying 1.3 kilobases of the 5' flanking region of the gene fused to the rat OCT cDNA was microinjected into fertilized eggs, and 17 transgenic mice were produced. Expression in the liver of the transgene was detected in three mice. In these mice, the transgene expression was observed exclusively in the liver and small intestine. Expression of the transgene in the intestine was comparable to that of the endogenous mouse OCT gene, whereas expression in the liver was much lower than that of the endogenous gene. The developmental pattern of expression of the transgene was similar to that of the endogenous gene. Therefore, the 5' flanking sequence of the rat OCT gene seems to be sufficient for the developmental and tissue-specific expression of the gene. An explanation for low expression in the liver remains the subject of ongoing study.     

In vivo pump‐probe optical coherence tomography imaging in Xenopus laevis

Currently, optical coherence tomography (OCT), is not capable of obtaining molecular information often crucial for identification of disease. To enable molecular imaging with OCT, we have further developed a technique that harnesses transient changes in light absorption in the sample to garner molecular information. A Fourier‐domain Pump‐Probe OCT (PPOCT) system utilizing a 532 nm pump and 830 nm probe has been developed for imaging hemoglobin. Methylene blue, a biological dye with well‐know photophysics, was used to characterize the system before investigating the origin of the hemoglobin PPOCT signal. The first in vivo PPOCT images were recorded of the vasculature in Xenopus laevis. The technique was shown to work equally well in flowing and nonflowing vessels. Furthermore, PPOCT was compared with other OCT extensions which require flow, such as Doppler OCT and phase‐variance OCT. PPOCT was shown to better delineate tortuous vessels, where nodes often restrict Doppler and phase‐variance reconstruction. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Noninvasive cross‐sectional imaging of proximal caries using swept‐source optical coherence tomography (SS‐OCT) in vivo

The aim of this study was to determine the diagnostic accuracy of swept‐source optical coherent tomography (SS‐OCT) in detecting and estimating the depth of proximal caries in posterior teeth in vivo. SS‐OCT images and bitewing radiographs were obtained from 86 proximal surfaces of 53 patients. Six examiners scored the locations according to a caries lesion depth scale (0–4) using SS‐OCT and the radiographs. The results were compared with clinical observations obtained after the treatment. SS‐OCT could detect the presence of proximal caries in tomograms that were synthesized based on the backscatter signal obtained from the proximal carious lesion through occlusal enamel. SS‐OCT showed significantly higher sensitivity and larger area under the receiver operating characteristic curve than radiographs for the detection of cavitated enamel lesions and dentin caries (Student's t ‐test, p < 0.05). SS‐OCT appears to be a more reliable and accurate method than bitewing radiographs for the detection and estimation of the depth of proximal lesions in the clinical environment. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Disruption of circulation by ethanol promotes fetal alcohol spectrum disorder (FASD) in medaka (Oryzias latipes) embryogenesis

Japanese medaka (Oryzias latipes) embryos exposed to ethanol have developed craniofacial, cardiovascular and skeletal defects which can be compared with the phenotypic features of fetal alcohol spectrum disorder (FASD) observed in human. The present experiment was designed to show that the disruption in circulation by ethanol during embryogenesis is a potential cause of FASD. Fertilized eggs were exposed to ethanol (0, 100 and/or 400 mM) for 24 or 48 h at various developmental stages (Iwamatsu stages 4-30) and were analyzed at 6 day post fertilization (dpf). It was observed that controls and the embryos exposed to 100 mM ethanol were in circulating state; however, a significant number of embryos of stages 4-24 exposed to 400 mM ethanol had disrupted circulation. Compared to controls, protein and RNA contents were significantly reduced in non-circulating embryos. Lipid peroxidation (LPO) analysis was made at 3, 6, 24, 48, 96 and 144 hour post fertilization (hpf). LPO was increased with the advancement of morphogenesis; however, ethanol or the circulation status had no effect. We further analyzed alcohol dehydrogenase (Adh 5 and adh8) and aldehyde dehydrogenase (Aldh9A and Aldh1A2) enzyme mRNAs in the embryos exposed to 400 mM ethanol for 24 h. A developmental stage-specific reduction in these enzyme mRNAs by ethanol was observed. We conclude that ethanol-induced disruption in circulation during embryogenesis is a potential cause of the development of FASD features in medaka.     

Quantifying dental biofilm growth using cross-polarization optical coherence tomography

Aims: Quantifying the ex vivo growth of complex multispecies dental biofilms using cross‐polarization 1310‐nm optical coherence tomography (CP‐OCT) system was investigated. Methods and Results: Bacterial microcosms, which were derived from plaque samples of paediatric subjects, were incubated in a biofilm reactor system containing discs of different dental materials for 72 h with daily sucrose pulsing (5×). CP‐OCT analysis of biofilm mass was validated with crystal violet (CV) assays at various growth stages of these complex biofilms. CP‐OCT was able to filter out the back‐reflected signals of water layers in the hydrated biofilm and allowed for direct biofilm quantification. The overall depth‐resolved scattering intensity of the biofilm showed very strong positive correlation with CV assay quantification (Spearman’s ρ = 0·92) during the growth phase of the biofilm. Conclusion: CP‐OCT was able to quantify the mass of the biofilm by measuring the overall depth‐resolved scattering of the biofilm. Significance and Impact of the Study: CP‐OCT has the ability to nondestructively monitor biofilm growth and elucidate the growth characteristics of these microcosms on different dental material compositions.     

High‐definition optical coherence tomography of melanocytic skin lesions

High‐definition optical coherence tomography (HD‐OCT) scanners have recently been developed. We assessed micromorphological HD‐OCT correlates of benign naevi (BN) and malignant melanoma (MM). 28 BN and 20 MM were studied using HD‐OCT and histology. Epidermal honeycomb/cobblestone pattern, regular junctional cell nests, and edged papillae are more often observed in BN, whereas fusion of rete ridges, pagetoid cells and junctional and/or dermal nests with atypical cells are more frequently seen in MM. A high overlap of HD‐OCT features in BN and MM was observed and in 20% of MM we did not find evidence for malignancy in OCT images at all. Using HD‐OCT it is possible to visualize architectural and cellular alterations of melanocytic skin lesions. The overlap of HD‐OCT features seen in BN and MM and the absence of suspicious HD‐OCT features in some MM represents an important limitation of HD‐OCT affecting the sensitivity of HD‐OCT in diagnosing MM.

High‐definition optical coherence tomography and the corresponding vertically sectioned histology of a compound naevus.