Synthesis of S-Carbamidomethyl Cysteine and Its Use for Quantification of Cysteinyl Peptides by Targeted Proteomics.

Proteomics has played a central role in the identification of reliable disease biomarkers, which are the basis of precision medicine, a promising approach for tackling recalcitrant diseases such as cancer, that elude conventional treatments. Among proteomic methodologies, targeted proteomics employing stable isotope-labeled (SIL) internal standards is particularly suited for the clinical translation of biomarker information owing to its high throughput and accuracy in the quantitative analysis of patient-derived proteomes. Using SIL internal standards ensures the utmost level of confidence in detection and precision in targeted MS experiments. For successfully establishing assays based on targeted proteomics, it is crucial to secure broad coverage when selecting the SIL standard peptide panel. However, cysteinyl peptides have often been excluded because of cysteine's high chemical reactivity. To address this limitation, a new cysteine building block was developed by incorporating a sulfhydryl group configured with an S-carbamidomethyl group, which is commonly used in proteome sampling. This compound was found to be chemically stable and applicable to a variety of solid-phase peptide synthesis (SPPS) campaigns. Furthermore, a direct comparison of the synthesized SIL peptides and tryptic endogenous peptides demonstrated the potential utility of an SPPS flow based on the new cysteine building block for improving the success of targeted proteomic applications.

Sex-Mediated Differences in TNF Signaling- and ECM-Related Gene Expression in Aged Rat Kidney.

Aging leads to the functional decline of an organism, which is associated with age and sex. To understand the functional change of kidneys depending on age and sex, we carried out a transcriptome analysis using RNA sequencing (RNA-Seq) data from rat kidneys. Four differentially expressed gene (DEG) sets were generated according to age and sex, and Gene Ontology analysis and overlapping analysis of Kyoto Encyclopedia of Genes and Genomes pathways were performed for the DEG sets. Through the analysis, we revealed that inflammation- and extracellular matrix (ECM)-related genes and pathways were upregulated in both males and females during aging, which was more prominent in old males than in old females. Furthermore, quantitative real-time PCR analysis confirmed that the expression of tumor necrosis factor (TNF) signaling-related genes, Birc3, Socs3, and Tnfrsf1b, and ECM-related genes, Cd44, Col3a1, and Col5a2, which showed that the genes were markedly upregulated in males and not females during aging. Also, hematoxylin-eosin (H&E) staining for histological analysis showed that renal damage was highly shown in old males rather than old females. In conclusion, in the rat kidney, the genes involved in TNF signaling and ECM accumulation are upregulated in males more than in females during aging. These results suggest that the upregulation of the genes may have a higher contribution to age-related kidney inflammation and fibrosis in males than in females.

Phase-Locked Synthetic Wavelength Interferometer Using a Femtosecond Laser for Absolute Distance Measurement without Cyclic Error.

We present a phase-locked synthetic wavelength interferometer that enables a complete elimination of cyclic errors in absolute distance measurements. With this method, the phase difference between the reference and measurement paths is fed back into a phase lock-in system, which is then used to control the synthetic wavelength and set the phase difference to zero using an external cavity acousto-optic modulator. We validated the cyclic error removal of the proposed phase-locked method by comparing it with the conventional phase-measuring method of the synthetic wavelength interferometer. By analyzing the locked error signal, we achieved a precision of 0.6 mrad in phase without any observed cyclic errors.

Open-Air Testing of Dual-Comb Time-of-Flight Measurement.

We configured a long-distance ranging apparatus to test the principle of dual-comb time-of-flight measurement using ultrashort lasers. Emphasis was given to the evaluation of open-air performance quantitatively in terms of the measurement resolution and stability. The test results revealed that our dual-comb asynchronous optical pulse sampling permits micrometer-resolved ranging with a repeatability of 2.05 µm over a 648 m distance in dry weather conditions. Further atmospheric effects were evaluated in three different weather conditions with corresponding Allan deviations. Finally, the capability of simultaneous determination of multiple targets was verified with the potential of advanced industrial applications, such as manufacturing, surveying, metrology, and geodesy.

Anti-Cancer Roles of Probiotic-Derived P8 Protein in Colorectal Cancer Cell Line DLD-1.

A novel probiotics-derived protein, P8, suppresses the growth of colorectal cancer (CRC). P8 can penetrate the cell membrane via endocytosis and cause cell cycle arrest in DLD-1 cells through down-regulation of CDK1/Cyclin B1. However, neither the protein involved in the endocytosis of P8 nor the cell cycle arrest targets of P8 are known. We identified two P8-interacting target proteins [importin subunit alpha-4 (KPNA3) and glycogen synthase kinase-3 beta (GSK3ß)] using P8 as a bait in pull-down assays of DLD-1 cell lysates. Endocytosed P8 in the cytosol was found to bind specifically to GSK3ß, preventing its inactivation by protein kinases AKT/CK1ε/PKA. The subsequent activation of GSK3ß led to strong phosphorylation (S33,37/T41) of ß-catenin, resulting in its subsequent degradation. P8 in the cytosol was also found to be translocated into the nucleus by KPNA3 and importin. In the nucleus, after its release, P8 binds directly to the intron regions of the GSK3ß gene, leading to dysregulation of GSK3ß transcription. GSK3ß is a key protein kinase in Wnt signaling, which controls cell proliferation during CRC development. P8 can result in a cell cycle arrest morphology in CRC cells, even when they are in the Wnt ON signaling state.

Regulation of Circadian Genes Nr1d1 and Nr1d2 in Sex-Different Manners during Liver Aging.

BACKGROUND: Circadian rhythm is associated with the aging process and sex differences; however, how age and sex can change circadian regulation systems remains unclear. Thus, we aimed to evaluate age- and sex-related changes in gene expression and identify sex-specific target molecules that can regulate aging. METHODS: Rat livers were categorized into four groups, namely, young male, old male, young female, and old female, and the expression of several genes involved in the regulation of the circadian rhythm was confirmed by in silico and in vitro studies. RESULTS: Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed that the expression of genes related to circadian rhythms changed more in males than in females during liver aging. In addition, differentially expressed gene analysis and quantitative real-time polymerase chain reaction/western blotting analysis revealed that Nr1d1 and Nr1d2 expression was upregulated in males during liver aging. Furthermore, the expression of other circadian genes, such as Arntl, Clock, Cry1/2, Per1/2, and Rora/c, decreased in males during liver aging; however, these genes showed various gene expression patterns in females during liver aging. CONCLUSIONS: Age-related elevation of Nr1d1/2 downregulates the expression of other circadian genes in males, but not females, during liver aging. Consequently, age-related upregulation of Nr1d1/2 may play a more crucial role in the change in circadian rhythms in males than in females during liver aging.

Osteopontin heptamer peptide containing the RGD motif enhances the phagocytic function of microglia.

Osteopontin (OPN) is a phosphorylated glycoprotein expressed in various tissues, including brain, and mediates a wide range of cellular activities. In our previous studies, we reported recombinant OPN and RGD and SLAY-containing OPN-peptide icosamer (OPNpt20) exhibited robust neuroprotective activities in an animal model of transient focal ischemia, and attributed these effects to the anti-inflammatory, pro-angiogenic, and phagocytic functions of OPNpt20. In the present study, we truncated OPNpt20 to 13 or 7 amino acid peptides containing RGD (R) and/or SLAY (S) motif (OPNpt13RS, OPNpt7R, OPNpt7RS, and OPNpt7S) and their cell motility and migration inducing activities were examined in BV2 cells (a microglia cell line). All four peptides significantly enhanced BV2 cell motility and migration, but OPNpt7R, an RGD-containing 7-amino-acid OPN peptide (VPNGRGD), was found to be most potent and its potency was comparable to OPNpt20. Phagocytic activity and F-actin polymerization were also significantly enhanced in OPNpt7R-treated BV2 cells. Importantly, studies using two mutant peptides (OPNpt7R-RAA and OPNpt7R-RAD, wherein RGD in OPNpt7R was replaced with RAA or RAD, respectively) revealed that all these effects of OPNpt7R, motility, migration, F-actin polymerization, and phagocytosis induction, were RGD-dependent. Furthermore, the Erk, Fak, and Akt signaling pathways appeared to be involved in the induction of phagocytic activity by OPNpt7R. Co-treating cells with OPNpt7R and D98059 or wortmannin (pharmacological inhibitors of Erk and Akt, respectively) significantly suppressed OPNpt7R-mediated phagocytosis induction. These results indicate the RGD-containing OPN heptamer OPNpt7R triggers microglial motility, migration, and phagocytic activity and that the RGD motif plays a critical role in these activities.

Absolute laser ranging by time-of-flight measurement of ultrashort light pulses [Invited].

Ultrashort pulse lasers are emerging as an advanced tool of distance measurement, with their unique temporal and spectral characteristics being extended to diverse principles of absolute ranging and instrumentation. Here, a systematic methodology is presented for absolute ranging by means of the time-of-flight measurement of ultrashort light pulses using dual-comb asynchronous optical sampling. Based on an elaborate uncertainty analysis, influencing system parameters such as the pulse duration, repetition rate, and averaging time are optimized to achieve a sub-µm measurement accuracy. The absolute ranging system developed in this study demonstrates a combined standard uncertainty of 0.986 µm for a 0.5 ms averaging over a distance range of 3.0 m, with a further reduction to 0.056 µm when the averaging time is increased to 0.5 s. The outstanding performance leads to unprecedented multitarget applications: machine feed control with thermal error compensation in real time as well as the nondestructive inspection of multilens assembly in a production line.

Diverging cyclic radial shearing interferometry for single-shot wavefront sensing.

In this investigation, we describe a simple cyclic radial shearing interferometer for single-shot wavefront sensing. Instead of using the telescope lens system used in typical radial shearing interferometry, a single lens is used to generate two diverging radial shearing beams. This simple modification leads to the advantages of conveniently adjusting the radial shearing ratio, compactness of the system, and practical ease of alignment. With the aid of a polarization pixelated CMOS camera, the spatial phase-shifting technique is used to extract the phase with a single image. The most important feature is the fringe contrast enhancement by reducing the aberrations caused by the complicated optical system even though an incoherent light is used. The experimental results show the fringe contrast enhancement is at least 0.1 better than that of the conventional method, and the wavefronts are properly reconstructed with less than 0.071λ root-mean-squared wavefront error regardless of the coherence of the light.

Improved Self-Calibration of a Multilateration System Based on Absolute Distance Measurement.

Multilateration tracking systems (MLTSs) are used in industrial three-dimensional (3D) coordinate measuring applications. For high-precision measurement, system parameters must be calibrated properly in advance. For an MLTS using absolute distance measurement (ADM), the conventional self-calibration method significantly reduces estimation efficiency because all system parameters are estimated simultaneously using a complicated residual function. This paper presents a novel self-calibration method that optimizes ADM to reduce the number of system parameters via highly precise and separate estimations of dead paths. Therefore, the residual function to estimate the tracking station locations can be simplified. By applying a suitable mathematical procedure and solving the initial guess problem without the aid of an external device, estimation accuracy of the system parameters is significantly improved. In three self-calibration experiments, with ADM repeatability of approximately 3.4 µm, the maximum deviation of the system parameters estimated by the proposed self-calibration method was 68.6 µm, while the maximum deviation estimated by the conventional self-calibration method was 711.9 µm. Validation of 3D coordinate measurements in a 1000 mm × 1000 mm × 1000 mm volume showed good agreement between the proposed ADM-based MLTS and a commercial laser tracker, where the maximum difference based on the standard deviation was 17.7 µm. Conversely, the maximum difference was 98.8 µm using the conventional self-calibration method. These results confirmed the efficiency and feasibility of the proposed self-calibration method.

Adenosine Triphosphate Accumulated Following Cerebral Ischemia Induces Neutrophil Extracellular Trap Formation.

In ischemic stroke, neutrophils infiltrate damaged brain tissue immediately following the ischemic insult and aggravate inflammation via various mechanisms which include neutrophil extracellular traps (NETs) formation. In the present study, we showed that adenosine triphosphate (ATP), a DAMP molecule, accumulates in the brain and induces NETosis in brain parenchyma and in circulating neutrophils (PMNs) isolated from a murine model of stroke induced by middle cerebral artery occlusion (MCAO). Expression of peptidylarginine deiminase-4 (PAD4), which induces citrullination of histones H3 (CitH3) and initiates NETosis, was significantly enhanced in brain parenchyma and blood PMNs following MCAO. ATP or BzATP (a prototypic P2X7R agonist) significantly enhanced the inductions of PAD4 and CitH3 in a P2X7R-dependent manner and intracellular Ca2+ influx, PKCα activation, and NADPH oxidase-dependent reactive oxygen species (ROS) production play critical roles in this ATP-P2X7R-mediated NETosis. In our MCAO animal model, NETosis was markedly suppressed by treatment with apyrase, an enzyme hydrolyzing ATP, but enhanced by co-treatment of BzATP, confirming ATP-P2X7R-mediated NETosis. Since ATP not only induced NETosis but was also extruded after NETosis, our results indicate that ATP accumulated in the ischemic brain induces NETosis, mediating a cross-talk linking NETosis with neuronal damage that might aggravate inflammation and brain damage.

Comb segmentation spectroscopy for rapid detection of molecular absorption lines.

We perform fast comb spectroscopy by dividing the probe comb into several sub-comb segments so as to produce multi-heterodyne beats focused around targeted molecular absorption lines. This concentrated scheme of comb spectroscopy is able to achieve a 30 dB signal-to-noise ratio with just a single shot measurement of 10 µs acquisition time. Such high signal sensitivity is verified by measuring separate absorption lines of H13C14N and 12CO2 gases simultaneously. In addition, atmospheric 12CO2 concentration over a 1.3 km open-air path is traced with a signal repeatability of 15 ppm at a 5 kHz update rate.

Rigorous single pulse imaging for ultrafast interferometric observation.

We discuss how to realize rigorous single pulse imaging using a fiber mode-locked laser for the purpose of ultrafast interferometric observation of fast varying dynamic objects. Sub-picosecond pulses are readily picked up in synchronization with the camera operation, allocating one pulse per frame, but rigorous ultrashort single pulse imaging is disturbed by the accumulation of amplified spontaneous emission (ASE) over the exposure time of the camera. Here, we propose four distinct methods to eliminate the ASE-accumulated disruption in the ultrashort optical gating by pulse interferometry and then evaluate their merits and limitations individually by experiments. The proposed four methods are referred to respectively as the time averaged phase modulation, unbalanced pulse overlapping, tandem pulse picking, and second harmonic generation.

Surface third-harmonic generation at a two-photon-polymerized micro-interferometer for real-time on-chip refractive index monitoring.

A micro-interferometer based on surface third-harmonic generation (THG) at two-photon-polymerized SU-8 cuboids for real-time monitoring of the refractive index changes of target fluids, which can be easily integrated into microfluidic photonic systems, is demonstrated. The third-harmonic (TH) interferogram is selectively generated only from the target volume by a simple vertical pumping, thereby eliminating the needs for complicated coupling and alignments. The dependence of the generated TH to the input pump polarization state is thoroughly investigated. The THG efficiency by linearly polarized excitation is found to be 2.6 × 10-7, which is the most efficient at the SU-8-air interface and independent of the input polarization direction. The THG efficiency from the SU-8-air interface is 12.17 times higher than that from the glass-air interface and 4.93 times higher than that from the SU-8-glass interface. Real-time monitoring of argon gas pressure is demonstrated using the micro- interferometer. The surface TH from two-photon-polymerized 3D structures offers novel design flexibility to the nonlinear optical light sources for microfluidic and microelectronic devices.

Anti-Zn2+-Toxicity of 4-Hydroxybenzyl Alcohol in Astrocytes and Neurons Contribute to a Robust Neuroprotective Effects in the Postischemic Brain.

4-Hydroxybenzyl alcohol (4-HBA) is an important phenolic constituent of Gastrodia elata (GE) Blume, which is used as a traditional herbal medicine in East Asia. Many activities have been reported to underlie the beneficial effects of 4-HBA in brain, such as, anti-oxidative, anti-inflammatory, anti-excitotoxic, and anti-apoptotic effects in neurons and microglia. Here, the authors demonstrate the robust neuroprotective effects of 4-HBA in rat middle cerebral artery occlusion (MCAO) model of stroke, and showed anti-Zn2+ toxicity in neurons and astrocytes as a molecular mechanism contributing to these effects. Intraperitoneal administration of 4-HBA (20 mg/kg) in Sprague-Dawley rats 1 h after MCAO reduced infarct volumes to 27.1 ± 9.2% of that of MCAO controls and significantly ameliorated motor impairments and neurological deficits. Significant suppressions of Zn2+-induced cell death, ROS generation, and PARP-1 induction by 4-HBA were observed in primary cortical cultures. 4-HBA also protected astrocytes from Zn2+-induced toxicity and suppressing ROS generation by employing slightly different molecular mechanisms, i.e., suppressing PARP-1 induction and NAD depletion under acute Zn2+-treatment and suppressing p67 NADPH oxidase subunit induction under chronic Zn2+-treatment. Results indicate that the protective effects of 4-HBA against Zn2+-toxicity in neurons and astrocytes contribute to its robust neuroprotective effects in the postischemic brain. Considering the pleiotropic effects of 4-HBA, which have been reported in previous reports and added in the present study, it has therapeutic potential for the amelioration of ischemic brain damage.

GSK-3ß-mediated regulation of cadmium-induced cell death and survival.

BACKGROUND: Previous studies indicated that cadmium (Cd) increases PI3-kinase/Akt phosphorylation, resulting in an alteration in GSK-3ß activity. However, the mechanism of Cd-induced endoplasmic reticulum (ER) stress in neuronal cells has yet to be studied in needs further elucidation. We examined the role of GSK-3ß in Cd-induced neuronal cell death and the related downstream signaling pathways. METHODS: SH-SY5Y human neuroblastoma cells were treated with 10 or 20 µM BAPTA-AM and 1 µM wortmannin for 30 min and then incubated with 25 µM Cd for 12 h. Apoptotic cells were visualized via DAPI and PI staining. Data were evaluated with one-way analysis of variance (ANOVA) followed by Student's t-test. Data are expressed as the means ± SD of experiments performed at least three times. RESULTS: Treatment of human neuronal SH-SY5Y cells with Cd induced ER, stress as evidenced by the increased expression of GRP78, which is a marker of ER stress. Cd exposure significantly increased the phosphorylation of Akt at thr308 and ser473 and that of GSK-3ß at ser9 in a time-dependent manner, while the total protein levels of GSK-3ß and Akt did not change. Cd-induced apoptosis was higher in GSK-3ß-knockdown cells than in normal cells. CONCLUSIONS: Our data suggest that Akt/GSK-3ß signaling activated by Cd is involved in neuronal cell survival.

3D profiling of rough silicon carbide surfaces by coherence scanning interferometry using a femtosecond laser.

We test an erbium-doped fiber femtosecond laser for its potential as a light source for a coherence scanning interferometer for large field-of-view profiling of rough silicon carbide (SiC) surfaces. This infrared fiber pulse laser is able to provide a relatively long temporal coherence length of ∼30 µm to be appropriate for coherence scanning of rough surfaces. At the same time, it offers a high degree of spatial coherence comparable to that of a monochromatic continuous wave laser to achieve a large measurement field of view. In addition, the highly maintained linear polarization of the pulse laser source permits overcoming the low specular reflectance of rough SiC surfaces by polarization-based optical power splitting control between the reference and measurement arms.

Large-aperture ground glass surface profile measurement using coherence scanning interferometry.

We present a coherence scanning interferometer configured to deal with rough glass surfaces exhibiting very low reflectance due to severe sub-surface light scattering. A compound light source is prepared by combining a superluminescent light-emitting diode with an ytterbium-doped fiber amplifier. The light source is attuned to offer a short temporal coherence length of 15 µm but with high spatial coherence to secure an adequate correlogram contrast by delivering strongly unbalanced optical power to the low reflectance target. In addition, the infrared spectral range of the light source is shifted close to the visible side at a 1,038 nm center wavelength, so a digital camera of multi-mega pixels available for industrial machine vision can be used to improve the correlogram contrast further with better lateral image resolutions. Experimental results obtained from a ground Zerodur mirror of 200 mm aperture size and 0.9 µm rms roughness are discussed to validate the proposed interferometer system.

Tuning range extension of pulse repetition rate using chirped fiber Bragg gratings.

We present a fiber femtosecond laser oscillator that incorporates a pair of chirped fiber Bragg gratings (CFBGs) to augment the cavity length elongation produced by a PZT actuator. With a magnification factor of 18.2, the CFBG pair offers a 4.44 mm cavity length extension, providing a 25.59 kHz tuning range of the repetition rate from a 41.56 MHz nominal value without loss of mode locking. The extended tuning range enhances the performance of comb stabilization in uncontrolled environments and pulse-to-pulse interferometry for step height measurements.

Nonlinear third harmonic generation at crystalline sapphires.

Third harmonic generation (THG) is a nonlinear optical phenomenon which can be applied in diverse research areas including interfacial studies, sub-wavelength light manipulation, and high sensitivity bio-molecular detection. Most precedent studies on THG have focused on dielectric and metallic materials, including silicon, gold, and germanium, due to their high nonlinear susceptibility. Sapphire, a widely-used optical substrate, has not been studied in depth for its third harmonic characteristics, despite its excellent optical transmission in the UV-visible range, high thermal conductance, and superior physical and chemical stability. In this research, we comprehensively studied THG at thin air-dielectric interfaces of sapphire wafers by controlling the wafer cutting planes, focusing depth, incidence angle, laser intensity, and input polarization of the input laser beam. These findings can lead to broader use of third harmonics for high-precision sapphire characterization, such as surface quality inspection, crystallinity determination, interfacial studies, delamination check, and real-time monitoring of crack propagation.