Integrated microRNA-mRNA analysis provides new insights into gonad coloration in the sea urchin Strongylocentrotus intermedius

Comparative microRNA (miRNA) and mRNA transcriptome analyses were performed on Strongylocentrotus intermedius of the same sex with significant gonadal color differences. The results indicated that 1) the color of female gonads was generally superior to that of males. 2) Comparative and integrated miRNA and mRNA transcriptome analyses identified differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) in female and male individuals with significant gonadal color differences. Common and sex-specific DEGs and “DEM-DEG” pairs involved in carotenoid absorption, accumulation, and transformation were identified as candidates correlated with gonad coloration in S. intermedius. Collectively, the results from this study have enriched our knowledge of the process of sea urchin gonad coloration and should provide additional clues for increasing the gonad quality of commercial sea urchins from molecular and metabolic aspects

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

Fiber-optic catheter for cellular resolution imaging

Patients with cystic fibrosis, primary ciliary dyskinesia and chronic obstructive lung disease suffer from airway narrowing and lung infection, leading to morbidity and mortality. Mucociliary clearance (MCC) is a critical self-defense mechanism that prevents respiratory system infection. Impairment of MCC is associated with alterations in the phenotype of epithelial ciliated, mucous, basal cells, etc. How- ever, further understanding of disease pathogenesis and corresponding development for disease diagnosis therapeutics are hindered by lack of an imaging tool for in vivo visualization of these altered phenotype of ciliary function and MCC in real time. Currently available imaging tools cannot provide comprehensive parameters of interest, periciliary liquid (PCL), airway surface liquid (ASL) depth, ciliary beat frequency (CBF) and mucociliary clearance transport (MCT) simultaneously. μOCT has been known as the highest resolution OCT technique available to date and validated as a powerful tool to evaluation MCC and ciliary function. The unprecedented resolution enables quantitative assessment of all parameters of in- terests at the same time within one imaging session. Several major developments were included in this dissertation to translate μOCT towards clinical applications and advance μOCT to higher resolution and better image quality. Four main topics were covered: 1. Visualization of cilia regeneration after injury. 2. Endoscopic μOCT fiber catheter design and prototype toward in vivo clinical applications. 3. Dual spectrometer for in vivo imaging of blood flow. 4. Multifiber μOCT catheter for speckle reduction. We first studied cilia in vivo regeneration with μOCT for a better understanding of the recovery process after certain diseases/accidents. We visualized adult mice tracheas up to 52 days after sulfur dioxide injury and quantified epithelial thickness, cilia coverage, length, beat frequency, coordination, and cilia-generated flow. We observed that regeneration of ciliated cells and cilia completed in 6 days but cilia gradually aligned and functioned in a coordinated way both on individual level and as a group for liquid transport. The later process takes longer. Notably, an overshoot in CBF was observed at the early stage when cilia just fully regenerated. It could be a sign of lag in cilia coordination at the early stage of regeneration. This study was described in Chapter 2. As the previous study was also limited to excised tissues, Chapter 3 presents a novel high-resolution endoscopic μOCT catheter, pushing the clinical feasibility of μOCT a big step forward. The μOCT catheter herein was designed and fabricated to perform longitudinal scanning in vivo in human airways. The outer diameter of 2.4 mm and flexibility allowed it to be inserted into the instrument channel of a standard bronchoscope. Under the real-time video guidance of the bronchoscope, the endoscopic fiber-optics catheter could reach the regions of interest up to sec- ondary bronchi and conduct high resolution, real time imaging of the functional microanatomy of the airway epithelium, including ciliary beat frequency and mu- cociliary transport rates, etc. We demonstrated the feasibility of the catheter using mouse trachea ex vivo and swine trachea in vivo. The next two chapters are dedicated to improve spatial resolution and image quality of μOCT based on benchtop systems. Chapter 4 presents a dual spectrometer μOCT system for extended spectrum band- width detection in order to achieve 1-μm axial resolution. The dual spectrometer design makes it possible to improve axial resolution using NIR radiation and gain improved penetration by use of longer center wavelength. The dual spectrometer utilized two line scan cameras of distinct detection wavelength ranges. The two spectral ranges were combined into a supercontinuum with extended full-width- half-maximum of 345 nm and thus the axial resolution in aqueous environment was 0.93 μm (n = 1.37). In vivo experiment showed blood flow in zebrafish larvae tail vein. Not only individual red blood cell was observed but also endothelial cells lining along the luminal surface of the blood vessel wall. Although at 1 μm resolution μOCT has enough resolving power to identity sub- cellular of biological tissue, some important microstructures was masked behind speckles. With the objective to improve imaging quality at high resolution, a mul- tifiber μOCT catheter was designed to reduce speckle, as described in Chapter 5. The key element in this design was a multi-facet fiber array which actively deliv- ered three light beams onto imaging sample and picked up back-scattered signal at different angles. Simultaneous multichannel spectrometer was employed to detect signals from different channels. Speckle reduction was realized by compounding signals from all channels. Rat esophagus imaging using this setup demonstrated an improved signal-to-noise ratio, contrast-to-noise ration and equivalent number of looks. The epithelium was better delineated from the overlying mucus and lamina propria beneath the basement membrane.Doctor of Philosoph

Analysis of special fibres for light generation

Suspended core fiber (SCF) is one of the key classes of microstructured optical fibers (MOFs), which incorporate three air holes within their cross-section. The holes within SCFs can offer significant overlap between guided mode of the fiber and the air holes and can be used to control the interactions between guided light and matter located within the holes. We report a silica optical fiber sensor using quantum dot (Qdot) filled Suspended Core Fiber (SCF). We explored the dipping sensing of SCF using shorter excitation wavelength and numerically and experimentally proved that shorter excitation wavelength gives a better fluorescence excitation and capture. In addition, we found a clearer distinction between glass fluorescence and Quantum Dot fluorescence which can reduce the crosstalk of them and increase the sensitivity of SCFs. Selective filling method to investigate the guidance performance concludes that filling all the holes in the SCF has the best outcome.Bachelor of Engineerin

A new method of online learning with kernels for regression

New optimization models and algorithms for online learning with kernels (OLK) in regression are proposed in a Reproducing Kernel Hilbert Space (RKHS) by solving a constrained optimization model. The “forgetting” factor in the model makes it possible that the memory requirement of the algorithm can be bounded as the learning process continues. The applications of the proposed OLK algorithms in classification and regression show their effectiveness in comparing with the state of art algorithms

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.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)NMRC (Natl Medical Research Council, S’pore

Depth extension and sidelobe suppression in optical coherence tomography using pupil filters

We demonstrate a new focus engineering scheme to achieve both extended depth of focus (DOF) and sidelobe suppression in spectral-domain optical coherence tomography (SD-OCT) system. Each of the illumination pupil function and the detection pupil function is modulated using an annular pupil filter implemented by center obscuration. The two pupil filters are arranged in a dark-field configuration such that the first sidelobe of the illumination point-spread function (PSF) matches the first minimum of the detection PSF in the lateral focal plane. We tested the feasibility of the proposed scheme numerically, and then constructed a dark-field OCT (DF-OCT) system to further verify its effectiveness experimentally. Simulation results show that a DOF gain of 4.2 can be achieved compared with a full aperture OCT (FA-OCT) system, with a suppression ratio of 2.9 dB for the first sidelobe compared with an annular-aperture bright-field OCT (BF-OCT) system. Experimental results show that the constructed DF-OCT extends the DOF by three-fold compared with the constructed FA-OCT, and suppresses the first sidelobe by 3.1 dB compared with the BF-OCT. The penalty for the extended DOF is an ~11.6 dB drop in sensitivity compared with the FA-OCT system.NRF (Natl Research Foundation, S’pore)Accepted versio

Characterization of a novel glutamate dehydrogenase gene and its response to heat stress in the sea urchin Strongylocentrotus intermedius

Glutamate dehydrogenase (GDH), a key metabolic enzyme that is ubiquitous across almost all living species, is essential for cell survival. To elucidate the characteristics and functions of the GDH gene in the sea urchin, we cloned and characterized the full-length cDNA of a novel GDH homolog from Strongylocentrotus intermedius, herein designated SiGDH. The full-length SiGDH gene was 2499 bp, with an open reading frame (ORF) of 1635 bp, encoding 544 amino acids. Bioinformatic analyses revealed that the predicted SiGDH protein contained the conserved ELFV_dehydrog_N and ELFV_dehydrog domains and that this protein had the highest sequence identity with the GDH protein from Strongylocentrotus purpuratus. Tissue-specific differences in SiGDH relative expression patterns and enzyme activity levels were observed, and the highest relative expression and total enzyme activity of SiGDH were determined to be in the gonad. Changes in SiGDH relative expression and enzyme activity in the gonad were observed after both gradual and acute heat stress. Together, our observations help to clarify the characteristics of this GDH homolog, as well as its associations with heat resistance in echinoderms

Effects of Different Doses of Fructooligosaccharides (FOS) on the Composition of Mice Fecal Microbiota, Especially the Bifidobacterium Composition

Fructooligosaccharides (FOS) are a well-known class of prebiotic and are considered to selectively stimulate the growth of bifidobacteria in the gut. Previous studies focused on the growth stimulation of Bifidobacterium, but they did not further investigate the bifidobacterial composition and the specific species that were stimulated. In this study, mice were fed with FOS in different doses for four weeks and the composition of fecal microbiota, in particular Bifidobacterium, was analyzed by sequencing the V3–V4 region and the groEL gene on the MiSeq platform, respectively. In the high-dose group, the relative abundance of Actinobacteria was significantly increased, which was mainly contributed by Bifidobacterium. At the genus level, the relative abundances of Blautia and Coprococcus were also significantly increased. Through the groEL sequencing, 14 species of Bifidobacterium were identified, among which B. pseudolongum was most abundant. After FOS treatment, B. pseudolongum became almost the sole bifidobacterial species (>95%). B. pseudolongum strains were isolated and demonstrated their ability to metabolize FOS by high performance liquid chromatography (HPLC). Therefore, we inferred that FOS significantly stimulated the growth of B. pseudolongum in mice. Further investigations are needed to reveal the mechanism of selectiveness between FOS and B. pseudolongum, which would aid our understanding of the basic principles between dietary carbohydrates and host health

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