Mucin-Type O-Glycosylation in the Drosophila Nervous System.

Mucin-type O-glycosylation, a predominant type of O-glycosylation, is an evolutionarily conserved posttranslational modification in animals. Mucin-type O-glycans are often found on mucins in the mucous membranes of the digestive tract. These glycan structures are also expressed in other cell types, such as blood cells and nephrocytes, and have crucial physiological functions. Altered expression of mucin-type O-glycans is known to be associated with several human disorders, including Tn syndrome and cancer; however, the physiological roles of mucin-type O-glycans in the mammalian brain remains largely unknown. The functions of mucin-type O-glycans have been studied in the fruit fly, Drosophila melanogaster. The basic structures of mucin-type O-glycans, including Tn antigen (GalNAcα1-Ser/Thr) and T antigen (Galß1-3GalNAcα1-Ser/Thr), as well as the glycosyltransferases that synthesize them, are conserved between Drosophila and mammals. These mucin-type O-glycans are expressed in the Drosophila nervous system, including the central nervous system (CNS) and neuromuscular junctions (NMJs). In primary cultured neurons of Drosophila, mucin-type O-glycans show a characteristic localization pattern in axons. Phenotypic analyses using mutants of glycosyltransferase genes have revealed that mucin-type O-glycans are required for CNS development, NMJ morphogenesis, and synaptic functions of NMJs in Drosophila. In this review, we describe the roles of mucin-type O-glycans in the Drosophila nervous system. These findings will provide insight into the functions of mucin-type O-glycans in the mammalian brain.

Glucuronylated core 1 glycans are required for precise localization of neuromuscular junctions and normal formation of basement membranes on Drosophila muscles.

T antigen (Galß1-3GalNAcα1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 ß1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including decreased number of circulating hemocytes, hyper-differentiation of hematopoietic stem cells in lymph glands, malformation of the central nervous system, mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAß1-3Galß1-3GalNAcα1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. In this study, for the first time, we unraveled biological roles of glucuronylated T antigen. Our data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila ß1,3-glucuronyltransferase-P (dGlcAT-P). We created dGlcAT-P null mutants and found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. We also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, we showed a genetic interaction between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization, basement membrane formation, and NMJ arborization on larval muscles.

Mucin-type core 1 glycans regulate the localization of neuromuscular junctions and establishment of muscle cell architecture in Drosophila.

T antigen (Galß1-3GalNAcα1-Ser/Thr), a core 1 mucin-type O-glycan structure, is synthesized by Drosophila core 1 ß1,3-galactosyltrasferase 1 (dC1GalT1) and is expressed in various tissues. We previously reported that dC1GalT1 synthesizes T antigen expressed in hemocytes, lymph glands, and the central nervous system (CNS) and that dC1GalT1 mutant larvae display decreased numbers of circulating hemocytes and excessive differentiation of hematopoietic stem cells in lymph glands. dC1GalT1 mutant larvae have also been shown to have morphological defects in the CNS. However, the functions of T antigen in other tissues remain largely unknown. In this study, we found that glycans contributed to the localization of neuromuscular junction (NMJ) boutons. In dC1GalT1 mutant larvae, NMJs were ectopically formed in the cleft between muscles 6 and 7 and connected with these two muscles. dC1GalT1 synthesized T antigen, which was expressed at NMJs. In addition, we determined the function of mucin-type O-glycans in muscle cells. In dC1GalT1 mutant muscles, myofibers and basement membranes were disorganized. Moreover, ultrastructural defects in NMJs and accumulation of large endosome-like structures within both NMJ boutons and muscle cells were observed in dC1GalT1 mutants. Taken together, these results demonstrated that mucin-type O-glycans synthesized by dC1GalT1 were involved in the localization of NMJ boutons, synaptogenesis of NMJs, establishment of muscle cell architecture, and endocytosis.

Label-free visualization of acetaminophen-induced liver injury by high-speed stimulated Raman scattering spectral microscopy and multivariate image analysis.

We recently established a high-speed, label-free, spectral imaging method based on stimulated Raman scattering (SRS). This method enables examination of cellular features within relatively short periods, thus enabling new imaging applications in pathology. Previously, we reported on label-free visualization of mouse tissue using SRS spectral microscopy combined with multivariate image analysis, but the feasibility of applying this approach to diseased tissues with diverse morphology and irregular chemical species has not been examined. We, therefore, assessed acetaminophen-induced liver injury to evaluate the potential use of Raman spectral microscopy for visualizing histopathologic specimens. Acetaminophen-overdosed mouse liver was prepared and the pathologic changes including centrilobular necrosis were confirmed. Multi-colored images were reconstructed through principal component analysis (PCA) of a multi-band SRS dataset, which provided rich information compared with a monochrome single-band SRS dataset. A wide view of the multi-colored principal component (PC) images showed the distribution of cellular constituents, which was similar to that observed by fat staining. In addition, different types of cells in liver parenchyma were also demonstrated. In conclusion, the combination of SRS spectral microscopy and PCA has the potential to reveal both the morphological and chemical features of specimens and therefore has potential utility in diagnostic pathology.

Pilot clinical study of Sasa senanensis Rehder leaf extract treatment on lichenoid dysplasia.

BACKGROUND: Previous studies have shown antiviral, antibacterial, and anti-inflammatory activity of alkaline extract of the leaves of Sasa senanensis Rehder (SE). Here, we investigated whether SE is effective on oral lichenoid dysplasia and osteoclastogenesis. MATERIALS AND METHODS: A male patient with white lacy streaks in the oral mucosa was orally administered SE three times a day for 11 months. The area of white streaks was monitored by intraoral photography. Interleukin-6 and -8 in the saliva were determined by enzyme-linked immunosorbent assay. Osteoclastogenesis of mouse macrophage-like RAW264.7 cells, induced by receptor activator of nuclear factor-κB ligand (RANKL) was monitored by tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cell formation. RESULTS: Long-term treatment with SE progressively reduced both the area of white steaks and the levels of salivary interleukin-6 and -8. SE significantly inhibited the macrophage differentiation towards osteoclasts. CONCLUSION: The present study suggests the therapeutic potential of SE towards oral diseases.

Sensitivity enhancement of fiber-laser-based stimulated Raman scattering microscopy by collinear balanced detection technique.

We propose the collinear balanced detection (CBD) technique for noise suppression in fiber laser (FL)-based stimulated Raman scattering (SRS) microscopy. This technique reduces the effect of laser intensity noise at a specific frequency by means of pulse splitting and recombination with a time delay difference. We experimentally confirm that CBD can suppress the intensity noise of second harmonic (SH) of Er-FL pulses by 13 dB.The measured noise level including the thermal noise is higher by only ~1.4 dB than the shot noise limit. To demonstrate SRS imaging, we use 4-ps SH pulses and 3-ps Yb-FL pulses, which are synchronized subharmonically with a jitter of 227 fs. The effectiveness of the CBD technique is confirmed through SRS imaging of a cultured HeLa cell.

Three-dimensional, non-invasive, cross-sectional imaging of protein crystals using ultrahigh resolution optical coherence tomography.

Micro-scale, non-invasive, three-dimensional cross-sectional imaging of protein crystals was successfully accomplished using ultra-high resolution optical coherence tomography (UHR-OCT) with low noise, Gaussian like supercontinuum. This technique facilitated visualization of protein crystals even those in medium that also contained substantial amounts of precipitates. We found the enhancement of the scattered signal from protein crystal by inclusion of agarose gel in the crystallization medium. Crystals of a protein and a salt in the same sample when visualized by UHR-OCT showed distinct physical characteristics, suggesting that protein and salt crystals may, in general, be distinguishable by UHR-OCT. UHR-OCT is a nondestructive and rapid method, which should therefore find use in automated systems designed to visualize crystals.

Stimulated Raman hyperspectral imaging based on spectral filtering of broadband fiber laser pulses.

We demonstrate a technique of hyperspectral imaging in stimulated Raman scattering (SRS) microscopy using a tunable optical filter, whose transmission wavelength can be varied quickly by a galvanometer mirror. Experimentally, broadband Yb fiber laser pulses are synchronized with picosecond Ti:sapphire pulses, and then spectrally filtered out by the filter. After amplification by fiber amplifiers, we obtain narrowband pulses with a spectral width of <3.3 cm(-1) and a wavelength tunability of >225 cm(-1). By using these pulses, we accomplish SRS imaging of polymer beads with spectral information.

Five-bit parallel operation of optical quantization and coding for photonic analog-to-digital conversion.

We report the attempt of optical quantization and coding in 5-bit parallel format for photonic A/D conversion. The proposed system is designed to realize generation of 32 different optical codes in proportion to the corresponding signal levels when fed a certain range of amplitude-varied input pulses to the setup. Optical coding in a bit-parallel format made it possible, that provides 5 bit optical codes from 32 optical quantized pulses. The 5-bit parallel operation of an optical quantization and coding module with 5 multi-ports was tested in our experimental setup.

Ultrashort pulse generation from continuous wave by pulse trapping in birefringent fibers.

We investigated the phenomenon of orthogonally polarized pulse trapping between a continuous-wave beam and an ultrashort soliton pulse in birefringent fibers both experimentally and numerically. Using pulse trapping and amplification, we demonstrated ultrashort pulse generation from a continuous-wave beam. The generated pulse had a nearly transform-limited sech(2)-shape and a temporal width of 350 fs. The obtained maximum pulse energy was 300 pJ using a 400 m-long low-birefringence fiber, and the corresponding gain was as large as 41 dB.

Quasi-supercontinuum generation using 1.06 µm ultrashort-pulse laser system for ultrahigh-resolution optical-coherence tomography.

We demonstrate quasi-supercontinuum (quasi-SC) generation around the 1.3 µm wavelength region using ultrahigh-speed, wavelength-tunable, femtosecond soliton pulses based on an ultrashort-pulse laser system operating at a wavelength of 1.0 µm. The wavelength tuning range was from 1.0 to 1.9 µm, and the scanning speed was up to 1.3 MHz. A Gaussian-like quasi-SC with a bandwidth of 220 nm was generated at 1220 nm. The generated quasi-SC was used in an optical-coherence tomography system. High axial resolutions of 5.1 µm in air and 3.7 µm in tissue were obtained. A maximum sensitivity of 100 dB was achieved, and ultrahigh-resolution images of a hamster cheek pouch were observed.

Stimulated Raman scattering microscope with shot noise limited sensitivity using subharmonically synchronized laser pulses.

We propose and demonstrate the use of subharmonically synchronized laser pulses for low-noise lock-in detection in stimulated Raman scattering (SRS) microscopy. In the experiment, Yb-fiber laser pulses at a repetition rate of 38 MHz are successfully synchronized to Ti:sapphire laser pulses at a repetition rate of 76 MHz with a jitter of <8 fs by a two-photon detector and an intra-cavity electro-optic modulator. By using these pulses, high-frequency lock-in detection of SRS signal is accomplished without high-speed optical modulation. The noise level of the lock-in signal is found to be higher than the shot noise limit only by 1.6 dB. We also demonstrate high-contrast, 3D imaging of unlabeled living cells.

Label-free imaging by stimulated parametric emission microscopy reveals a difference in hemoglobin distribution between live and fixed erythrocytes.

We demonstrate that stimulated parametric emission (SPE) microscopy enables label-free, 3-D visualization of internal hemoglobin distribution of live mouse and chicken erythrocytes with high sensitivity. Change in hemoglobin distribution in chicken erythrocytes before and after ethanol fixation is clearly visualized.

High-speed three-dimensional measurement using electronically controlled wavelength-tunable ultrashort pulse fiber laser.

A high-speed three-dimensional measurement system was demonstrated using an electronically controlled wavelength-tunable ultrashort pulse fiber laser and an all-fiber interferometer. A longitudinal scanning range of 2.8 mm and a scanning speed of 100,000 points/s were achieved without moving parts. For a measurement distance of 0.7 m, the sensitivity and accuracy were 70 dB and +/-8 microm, respectively. A clear three-dimensional image of a 100 yen Japanese coin was obtained using this measurement system.

Analysis and experimental assessment of the sensitivity of stimulated Raman scattering microscopy.

We theoretically show that the shot-noise-limited sensitivity of stimulated Raman scattering (SRS) microscopy, which enables high-contrast vibrational imaging, is similar to that of coherent anti-Stokes Raman scattering microscopy. We experimentally confirm that the sensitivity of our SRS microscope is lower than the shot-noise limit only by <15 dB, which indicates that the high-sensitivity of SRS microscopy is readily available.

Simultaneous amplitude and phase modulation by a discrete phase-only filter.

We propose a simultaneous amplitude and phase modulation method by a discrete phase-only filter. The proposed amplitude-phase filter can be realized by a discrete phase modulation of the diffractive optical element as well as a continuous phase modulation of the liquid crystal spatial light modulator. The fabricated amplitude-phase filter that has the six phase modulation levels shows a transfer efficiency of 75% regardless of the polarization state of the incident light. By using the proposed amplitude-phase filter, we demonstrate a temporal waveform conversion from sech(2) to super-Gaussian, which requires both amplitude and phase modulations.

In vivo manipulation of fluorescently labeled organelles in living cells by multiphoton excitation.

Femtosecond laser pulses in the near-infrared region have potential applications in the imaging and manipulation of intracellular organelles. We report on the manipulation of intracellular organelles by two-photon excitation. The dynamics of green fluorescent protein (GFP)-histone are investigated by two-photon fluorescence recovery after photobleaching (FRAP). Intracellular ablation of fluorescently labeled organelles in living cells is performed by focusing femtosecond laser pulses. We report on the selective marking of individual organelles by using two-photon conversion of a photoconvertible fluorescent protein.

Broadband group delay dispersion compensation for a microscope objective lens with a specially-designed mechanical deformable mirror.

We propose that the bending profile of a mechanical deformable mirror can be designed by shaping its form, realizing a simple, compact, and broadband group delay dispersion compensator in a 4-f pulse shaper arrangement. By using the proposed compensator, spectral phase distortion of a microscope objective lens is successfully pre-compensated for to generate a sub-8 fs pulse at the focus of the lens.

Highly-sensitive and high-resolution all-fiber three-dimensional measurement system.

A practical all-fiber three-dimensional measurement system is demonstrated with an incoherent interferometer at the eye-safe wavelength of 1.55 mum. The sensitivity and axial resolution are as high as 102 dB and 1.4 mum from a few meters' distance, respectively. A rotating scanner is developed for axial scanning, and a wide longitudinal scanning range of 54 mm is demonstrated. The high resolution images of a few samples are clearly obtained at the speed of 52 points/s. Moreover, the resolution, sensitivity, speed, and angle dependence are discussed for measurement of a 100 yen Japanese coin.

Deterministic design of binary phase-only blazed grating with subwavelength features under limitation on spatial resolution of fabrication technique.

We propose a deterministic design method of a blazed grating consisting of a binary grating with subwavelength structures for a multilevel phase modulation. The feasible shapes of binary subwavelength microstructures are restricted to a few kinds of surface profiles by constraints in an actual fabrication technique. The relationship between the feasible shapes of binary subwavelength microstructures and their phase modulations can be calculated by an electromagnetic analysis and tabulated. Using the relationship, a deterministic design of a binary grating with subwavelength structures is simply realized. We have designed the binary blazed grating with subwavelength structures and investigated its performance. Its diffraction efficiency is in good agreement with that by a conventional statistical design method.