Interactive effects of chronic ocean acidification and warming on the growth, survival, and physiological responses of adults of the temperate sea urchin Strongylocentrotusintermedius.

To investigate the interactive effects of chronic ocean acidification and warming (OAW) on the growth, survival, and physiological responses of sea urchins, adults of the temperate sea urchin Strongylocentrotus intermedius were incubated separately/jointly in acidic (ΔpHNBS = -0.5 units) and thermal (ΔT = +3.0 °C) seawater for 120 days under lab-controlled conditions based on the projected ocean pH and temperature for 2100 put forward by the Intergovernmental Panel on Climate Change (IPCC). Survival rate (SR), average food consumption rate (FCR), gut index (GuI), specific growth rate (SGR), digestive capability, energy production, and antioxidant capability were subsequently determined. The results showed that 1) the SR, FCR, GuI and SGR decreased sharply under OAW conditions. Significant interactive effects of OAW on SR and SGR were observed at 120 days post-incubation (dpi), and on FCR this occurred at 90 dpi. 2) OAW altered the activities of both digestive and antioxidant enzymes. There were significant interaction effects of OAW on the activities of amylase, trehalase, and superoxide dismutase. 3) The relative gene expression levels and activities of key enzymes involved in glycometabolism pathways (i.e., glycolysis and the tricarboxylic acid cycle) were significantly affected by OAW, resulting in an alteration of the total ATP content in the sea urchins. Interaction effects of OAW were observed in both relative gene expression and the activity of enzymes involved in glycolysis (hexokinase), the transformation of glycolysis end-products (lactate dehydrogenase), the tricarboxylic acid cycle (citrate synthetase), and ATP production (Na+/K+-ATPase). The data from this study will enrich our knowledge concerning the combined effects of global climate change on the survival, growth, and physiological responses of echinoderms.

miRn-3 inhibits cutaneous wound healing by targeting gelsolin in the sea cucumber Apostichopus japonicus.

The microRNA novel-3 (miRn-3) is a 23-nt small endogenous noncoding RNA of unknown function. To enrich our knowledge of the regulatory function of miRn-3 in the process of wound healing, the sea cucumber Apostichopus japonicus was used as a target model in this study. Gelsolin (AjGSN), a potential target gene of miRn-3, was cloned and characterized, and the interaction between miRn-3 and AjGSN was verified. The function of the miRn-3/AjGSN axis in regulating cutaneous wound healing was explored in the sea cucumber A. japonicus. The results showed that 1) the full-length cDNA of AjGSN was 2935 bp, with a high level of sequence conservation across the echinoderms; 2) miRn-3 could bind to the 3'UTR of AjGSN and negatively regulate the expression of AjGSN; 3) overexpression of miRn-3 and inhibition of the expression of AjGSN suppressed cutaneous wound healing in A. japonicus. In general, all observations of this study suggest that miRn-3 plays an important role in the early process of cutaneous wound healing by negatively targeting AjGSN, and that it may be a potential biomarker in wound healing.

Responses of sea urchins (Strongylocentrotus intermedius) with different sexes to CO2-induced seawater acidification: Histology, physiology, and metabolomics.

Responses of different sexes of farmed Strongylocentrotus intermedius to chronic CO2-induced seawater acidification were investigated in 120-day lab-based experiments. Four experimental groups were set up as one control group and three seawater acidification groups. The results showed that 1) Specific growth rate and the numbers of mature gamete cells declined in a pH-dependent way in both sexes of adult S. intermedius. 2) There were differences in SDMs identified in females and males reared in acidified seawater reflecting sex-specific response variation in adult S. intermedius. 3) The number of altered metabolic pathways exhibited a linear increasing trend as seawater pH declined in both sexes of adult S. intermedius. Meanwhile seawater acidification might affect metabolic processes via changing the relative expression and activity of key enzymes controlling the corresponding metabolic pathways of adult S. intermedius.

Distant hybrids of Heliocidaris crassispina (♀) and Strongylocentrotus intermedius (♂): identification and mtDNA heteroplasmy analysis.

BACKGROUND: Distant hybridization between the sea urchin Heliocidaris crassispina (♀) and the sea urchin Strongylocentrotus intermedius (♂) was successfully performed under laboratory conditions. A new variety of hybrid sea urchin (HS hybrid) was obtained. However, the early-development success rates for the HS hybrids were significantly lower than those of purebred H. crassispina or S. intermedius offspring. In addition, it was difficult to distinguish the HS-hybrid adults from the pure H. crassispina adults, which might lead to confusion in subsequent breeding attempts. In this study, we attempted to develop a method to quickly and effectively identify HS hybrids, and to preliminarily investigate the molecular mechanisms underlying the poor early-development success rates in the HS hybrids. RESULTS: The hybrid sea urchins (HS hybrids) were identified both morphologically and molecularly. There were no significant differences in the test height to test diameter ratios between the HS hybrids and the parents. The number and arrangement of ambulacral pore pairs in the HS hybrids differed from those of the parental lines, which might serve as a useful morphological character for the identification of the HS hybrids. A primer pair that identified the HS hybrids was screened by comparing the mitochondrial genomes of the parental lines. Moreover, paternal leakage induced mitochondrial DNA heteroplasmy in the HS hybrids, which might explain the low rates of early development success in these hybrids. CONCLUSIONS: The distant-hybrid sea urchins were accurately identified using comparative morphological and molecular genetic methods. The first evidence of mtDNA heteroplasmy after the distant hybridization of an echinoderm was also provided.

Comparative metabolome analysis provides new insights into increased larval mortality under seawater acidification in the sea urchin Strongylocentrotus intermedius.

Mortality and metabolic responses of four-armed larvae of Strongylocentrotus intermedius under CO2-induced seawater acidification were investigated. Gametes of S. intermedius were fertilized and developed to the four-armed larval stage in either current natural seawater pH levels (as Control; pH = 7.99 ± 0.01) or laboratory-controlled acidified conditions (OA1: ΔpH = -0.3 units; OA2: ΔpH = -0.4 units; OA3: ΔpH = -0.5 units) according to the predictions of the Intergovernmental Panel on Climate Change (IPCC). The degrees of spicule exposure and asymmetry and mortality of four-armed larvae of S. intermedius were observed; each had a significant linearly increasing trend as the seawater pH level decreased. Comparative metabolome analysis identified a total of 87 significantly differentially expressed metabolites (SDMs, UP: 57, DOWN: 30) in OA-treated groups compared with the control group. Twenty-three SDMs, including carnitine, lysophosphatidylcholine (LPC) 18:3, lysophosphatidyl ethanolamine (LPE) 16:1, glutathione (GSH) and L-ascorbate, exhibited a linear increasing trend with decreasing seawater pH. Nine SDMs exhibited a linear decreasing trend as the seawater pH declined, including hypoxanthine, guanine and thymidine. Among all SDMs, we further mined 48 potential metabolite biomarkers responding to seawater acidification in four-armed larvae of S. intermedius. These potential metabolite biomarkers were mainly enriched in five pathways: glycerophospholipid metabolism, glutathione metabolism, purine metabolism, pyrimidine metabolism and the tricarboxylic acid cycle (TCA cycle). Our results will enrich our knowledge of the molecular mechanisms employed by sea urchins in response to CO2-induced seawater acidification.

CO2-driven ocean acidification repressed the growth of adult sea urchin Strongylocentrotus intermedius by impairing intestine function.

Strongylocentrotus intermedius cultured in the northern Yellow Sea in China was utilized to evaluate the effects of chronic CO2-driven ocean acidification (OA) on adult sea urchins. Based on the projection of the Intergovernmental Panel on Climate Change (IPCC), present natural seawater conditions (pHNBS = 8.10 ± 0.03) and three laboratory-controlled OA conditions (OA1, ΔpHNBS = - 0.3 units; OA2, ΔpHNBS = - 0.4 units; OA3, ΔpHNBS = - 0.5 units) were employed. After 60-day incubation, our results showed that (1) OA significantly repressed the growth of adult S. intermedius; (2) food consumption tended to be decreased with pH decline; (3) intestinal morphology was changed, and activities of intestinal cellulase and lipase were decreased under acidified conditions; (4) expression levels of two immune-related genes (SiTNF14 and SiTGF-ß) were altered; (5) rate-limiting enzyme activities of the glycolytic pathway and tricarboxylic acid cycle (TAC) were changed in all OA treatments compared to those of controls.

MicroRNAs involved in innate immunity regulation in the sea cucumber: A review.

The sea cucumber is one of the most economically significant echinoderms. The immunity against exogenous stimulation of sea cucumber is of great academic and economic importance. MicroRNAs (miRNAs) are a class of short endogenous non-coding RNAs (ncRNAs) that are considered as vital regulators of both innate and adaptive immune responses in most eukaryotes. In sea cucumbers, some miRNAs (such as miR-133, miR-137, and miR-2008, among others) that participate in the regulation of innate immunity have been recently identified and characterized. This review focuses on those known miRNAs and their corresponding target genes that participate in the regulation of the complement system, Toll-like receptor (TLR) pathway, reactive oxygen species (ROS) production and apoptosis pathways in sea cucumbers. Moreover, we cover immune-related miRNA investigations in sea cucumbers that provide insights into developing more miRNA-based biomarkers and therapeutic strategies for sea cucumber diseases.

Intranasal micro-optical coherence tomography imaging for cystic fibrosis studies.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Although impairment of mucociliary clearance contributes to severe morbidity and mortality in people with CF, a clear understanding of the pathophysiology is lacking. This is, in part, due to the absence of clinical imaging techniques capable of capturing CFTR-dependent functional metrics at the cellular level. Here, we report the clinical translation of a 1-µm resolution micro-optical coherence tomography (µOCT) technology to quantitatively characterize the functional microanatomy of human upper airways. Using a minimally invasive intranasal imaging approach, we performed a clinical study on age- and sex-matched CF and control groups. We observed delayed mucociliary transport rate at the cellular level, depletion of periciliary liquid layer, and prevalent loss of ciliation in subjects with CF. Distinctive morphological differences in mucus and various forms of epithelial injury were also revealed by µOCT imaging and had prominent effects on the mucociliary transport apparatus. Elevated mucus reflectance intensity in CF, a proxy for viscosity in situ, had a dominant effect. These results demonstrate the utility of µOCT to determine epithelial function and monitor disease status of CF airways on a per-patient basis, with applicability for other diseases of mucus clearance.

Comparative transcriptome analysis identifies genes associated with papilla development in the sea cucumber Apostichopus japonicus.

To explore the molecular mechanisms underlying the initial differentiation and formation of papillae in sea cucumbers, the transcriptomes of Apostichopus japonicus pentactulae (without papillae) were compared to those of A. japonicus juveniles (with papillae). From the RNA of the three pentactula libraries and the three juvenile libraries, we obtained 41-46 million raw reads, yielding 39-45 million clean reads. From these, we generated 599,673 transcripts and identified 230,604 unigenes. Across all six transcriptomes, we identified 246,207 single nucleotide polymorphisms (SNPs) and 80,455 single sequence repeats (SSRs). There were more transition SNPs (60.74%) than transversion SNPs (39.26%). The mononucleotide repeat was the most abundant SSR motif. We identified 7965 differentially expressed unigenes (DEGs) in the juveniles and the pentactulae; 2421 DEGs were expressed only in the juveniles, and 1238 were expressed only in the pentactulae. Of all DEGs, 5215 were significantly upregulated and 2750 were significantly downregulated in the juveniles as compared to the pentactulae. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that the DEGs were primarily enriched in ribosome synthesis, cell proliferation, tight junction formation, collagen fibrillogenesis, and neurogenesis.

Identification and characterization of microRNAs from the tube foot in the sea urchin Strongylocentrotus intermedius.

MicroRNAs (miRNAs) play critical roles in regulating many bio-processes of eukaryotes. The sea urchin Strongylocentrotus intermedius (an important fishery resource) is of great economic importance in Japan, North Korea, Russia, and China. In the current study, miRNAs of tube foot in S. intermedius were firstly identified and characterized. Data in this study can provide more genomic information for the further understanding of the complex regulation network in sea urchins and present a new way for monitoring the health status of cultured sea urchins.

Endomicroscopic optical coherence tomography for cellular resolution imaging of gastrointestinal tracts.

Our ability to detect neoplastic changes in gastrointestinal (GI) tracts is limited by the lack of an endomicroscopic imaging tool that provides cellular-level structural details of GI mucosa over a large tissue area. In this article, we report a fiber-optic-based micro-optical coherence tomography (µOCT) system and demonstrate its capability to acquire cellular-level details of GI tissue through circumferential scanning. The system achieves an axial resolution of 2.48 µm in air and a transverse resolution of 4.8 µm with a depth-of-focus (DOF) of ~150 µm. To mitigate the issue of limited DOF, we used a rigid sheath to maintain a circular lumen and center the distal-end optics. The sensitivity is tested to be 98.8 dB with an illumination power of 15.6 mW on the sample. With fresh swine colon tissues imaged ex vivo, detailed structures such as crypt lumens and goblet cells can be clearly resolved, demonstrating that this fiber-optic µOCT system is capable of visualizing cellular-level morphological features. We also demonstrate that time-lapsed frame averaging and imaging speckle reduction are essential for clearly visualizing cellular-level details. Further development of a clinically viable µOCT endomicroscope is likely to improve the diagnostic outcome of GI cancers.

Development of a Primary Human Co-Culture Model of Inflamed Airway Mucosa.

Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 µm pore-sized transwells, compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production, consistent with conventional ALIs, as visualized by micro-optical coherence tomography (µOCT). µOCT is a recently developed imaging modality with the capacity for real time two- and three-dimensional analysis of cellular events in marked detail, including neutrophil transmigratory dynamics. Further, the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients, and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with µOCT imaging offers significant opportunity to probe, in great detail, micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface.

Illuminating dynamic neutrophil trans-epithelial migration with micro-optical coherence tomography.

A model of neutrophil migration across epithelia is desirable to interrogate the underlying mechanisms of neutrophilic breach of mucosal barriers. A co-culture system consisting of a polarized mucosal epithelium and human neutrophils can provide a versatile model of trans-epithelial migration in vitro, but observations are typically limited to quantification of migrated neutrophils by myeloperoxidase correlation, a destructive assay that precludes direct longitudinal study. Our laboratory has recently developed a new isotropic 1-µm resolution optical imaging technique termed micro-optical coherence tomography (µOCT) that enables 4D (x,y,z,t) visualization of neutrophils in the co-culture environment. By applying µOCT to the trans-epithelial migration model, we can robustly monitor the spatial distribution as well as the quantity of neutrophils chemotactically crossing the epithelial boundary over time. Here, we demonstrate the imaging and quantitative migration results of our system as applied to neutrophils migrating across intestinal epithelia in response to a chemoattractant. We also demonstrate that perturbation of a key molecular event known to be critical for effective neutrophil trans-epithelial migration (CD18 engagement) substantially impacts this process both qualitatively and quantitatively.

Flexible, high-resolution micro-optical coherence tomography endobronchial probe toward in vivo imaging of cilia.

We report the design and fabrication of a flexible, longitudinally scanning high-resolution micro-optical coherence tomography (µOCT) endobronchial probe, optimized for micro-anatomical imaging in airways. The 2.4 mm diameter and flexibility of the probe allows it to be inserted into the instrument channel of a standard bronchoscope, enabling real-time video guidance of probe placement. To generate a depth-of-focus enhancing annular beam, we utilized a new fabrication method, whereby a hollow glass ferrule was angle-polished and gold-coated to produce an elongated annular reflector. We present validation data that verifies the preservation of linear scanning, despite the use of flexible materials. When utilized on excised, cultured mouse trachea, the probe acquired images of comparable quality to those obtained by a benchtop µOCT system.

Single-camera full-range high-resolution spectral domain optical coherence tomography.

We developed spectral domain optical coherence tomography using a dual-channel spectrometer for complex conjugate artifacts (CCA) suppression. We used a three-line charge coupled device to simultaneously detect two interferometric spectra with 2π/3 phase difference. The complex interferometric signal was reconstructed by trigonometric manipulation of two real interferometric spectra, and then full-range images were obtained by use of inverse Fourier transform. Artifacts at direct current (DC) and the ghost remnant of the CCA are common issues with the previously reported two-spectrometer method because the slight mismatching between two spectral detection channels had strong negative effects on CCA suppression and appeared to be the limiting factor on system performance. This novel dual-channel spectrometer uses the same spectrometer optics for the two spectral detection channels and, therefore, achieves better matching between two spectral detection channels and consequently better performance in CCA suppression as compared with the dual spectrometer solution. Full-range imaging with CCA suppression up to ∼25 dB was demonstrated when imaging an attenuated reflector. The efficacy of both CCA and DC suppressions also was validated by imaging the anterior segment of a rat eye ex vivo. The quality of CCA-suppressed images was significantly improved with regard to those obtained with the dual-spectrometer design.

Multifiber angular compounding optical coherence tomography for speckle reduction.

We report on an integrated fiber optic design to implement multifiber angular compounding optical coherence tomography, which enables angular compounding for speckle reduction. A multi-facet fiber array delivers three light beams to the sample with different incident angles. Back-reflective/back-scattered signals from these channels were simultaneously detected by a three-channel spectrometer. The axial and lateral resolution was measured to be ∼3 and ∼3.5 µm, respectively, in air with ∼100 dB sensitivity. We conducted ex vivo experiments on a rat esophagus to demonstrate a contrast to noise improvement of 1.58.

In vivo imaging of airway cilia and mucus clearance with micro-optical coherence tomography.

We have designed and fabricated a 4 mm diameter rigid endoscopic probe to obtain high resolution micro-optical coherence tomography (µOCT) images from the tracheal epithelium of living swine. Our common-path fiber-optic probe used gradient-index focusing optics, a selectively coated prism reflector to implement a circular-obscuration apodization for depth-of-focus enhancement, and a common-path reference arm and an ultra-broadbrand supercontinuum laser to achieve high axial resolution. Benchtop characterization demonstrated lateral and axial resolutions of 3.4 µm and 1.7 µm, respectively (in tissue). Mechanical standoff rails flanking the imaging window allowed the epithelial surface to be maintained in focus without disrupting mucus flow. During in vivo imaging, relative motion was mitigated by inflating an airway balloon to hold the standoff rails on the epithelium. Software implemented image stabilization was also implemented during post-processing. The resulting image sequences yielded co-registered quantitative outputs of airway surface liquid and periciliary liquid layer thicknesses, ciliary beat frequency, and mucociliary transport rate, metrics that directly indicate airway epithelial function that have dominated in vitro research in diseases such as cystic fibrosis, but have not been available in vivo.

Modeling of Mechanical Stress Exerted by Cholesterol Crystallization on Atherosclerotic Plaques.

Plaque rupture is the critical cause of cardiovascular thrombosis, but the detailed mechanisms are not fully understood. Recent studies have found abundant cholesterol crystals in ruptured plaques, and it has been proposed that the rapid expansion of cholesterol crystals in a limited space during crystallization may contribute to plaque rupture. To evaluate the effect of cholesterol crystal growth on atherosclerotic plaques, we modeled the expansion of cholesterol crystals during the crystallization process in the necrotic core and estimated the stress on the thin cap with different arrangements of cholesterol crystals. We developed a two-dimensional finite element method model of atherosclerotic plaques containing expanding cholesterol crystals and investigated the effect of the magnitude and distribution of crystallization on the peak circumferential stress born by the cap. Using micro-optical coherence tomography (µOCT), we extracted the cross-sectional geometric information of cholesterol crystals in human atherosclerotic aorta tissue ex vivo and applied the information to the model. The results demonstrate that (1) the peak circumference stress is proportionally dependent on the cholesterol crystal growth; (2) cholesterol crystals at the cap shoulder impose the highest peak circumference stress; and (3) spatial distributions of cholesterol crystals have a significant impact on the peak circumference stress: evenly distributed cholesterol crystals exert less peak circumferential stress on the cap than concentrated crystals.

Spectral estimation optical coherence tomography for axial super-resolution.

The depth reflectivity profile of Fourier domain optical coherence tomography (FD-OCT) is estimated from the inverse Fourier transform of the spectral interference signals (interferograms). As a result, the axial resolution is fundamentally limited by the coherence length of the light source. We demonstrate that using the autoregressive spectral estimation technique instead of the inverse Fourier transform, to analyze the spectral interferograms can improve the axial resolution. We name this method spectral estimation OCT (SE-OCT). SE-OCT breaks the coherence length limitation and improves the axial resolution by a factor of up to 4.7 compared with FD-OCT. Furthermore, SE-OCT provides complete sidelobe suppression in the depth point-spread function, further improving the image quality. We demonstrate that these technical advances enables clear identification of corneal endothelium anatomical details ex vivo that cannot be identified using the corresponding FD-OCT. Given that SE-OCT can be implemented in the FD-OCT devices without any hardware changes, the new capabilities provided by SE-OCT are likely to offer immediate improvements to the diagnosis and management of diseases based on OCT imaging.

Spectrally encoded extended source optical coherence tomography.

We have developed an extended source optical coherence tomography (SEES-OCT) technique in an attempt to improve signal strength for ophthalmic imaging. A line illumination with a visual angle of 7.9 mrad is produced by introducing a dispersive element in the infinity space of the sample arm. The maximum permissible exposure (MPE) of such an extended source is 3.1 times larger than that of a "standard" point source OCT, which corresponds to sensitivity improvement of 5 dB. The advantage of SEES-OCT in providing superior penetration depth over a point source system is demonstrated using swine eye tissues ex vivo.