Large-scale analysis of the N-terminal regulatory elements of the kinase domain in plant Receptor-like kinase family.

BACKGROUND: The N-terminal regulatory element (NRE) of Receptor-like kinases (RLKs), consisting of the juxtamembrane segment in receptor kinases (RKs) and the N-terminal extension segment in RLCKs, is a crucial component that regulates the activities of these proteins. However, the features and functions of the NRE have remained largely unexplored. Herein, we comprehensively analyze 510,233 NRE sequences in RLKs from 528 plant species, using information theory and data mining techniques to unravel their common characteristics and diversity. We also use recombinant RKs to investigate the function of the NRE in vitro. RESULTS: Our findings indicate that the majority of NRE segments are around 40-80 amino acids in length and feature a serine-rich region and a 14-amino-acid consensus sequence, 'FSYEELEKAT[D/N]NF[S/D]', which contains a characteristic α-helix and ST motif that connects to the core kinase domain. This conserved signature sequence is capable of suppressing FERONIA's kinase activity. A motif discovery algorithm identifies 29 motifs with highly conserved phosphorylation sites in RK and RLCK classes, especially the motif 'VGPWKpTGLpSGQLQKAFVTGVP' in LRR-VI-2 class. Phosphorylation of an NRE motif in an LRR-VI-2 member, MDIS1, modulates the auto-phosphorylation of its co-receptor, MIK1, indicating the potential role of NRE as a 'kinase switch' in RLK activation. Furthermore, the characterization of phosphorylatable NRE motifs improves the accuracy of predicting phosphorylatable sites. CONCLUSIONS: Our study provides a comprehensive dataset to investigate NRE segments from individual RLKs and enhances our understanding of the underlying mechanisms of RLK signal transduction and kinase activation processes in plant adaptation.

Morphological and functional characteristics of aging kidneys based on two-photon microscopy in vivo.

Age-related kidney disease, which is chronic and naturally occurring, is a general term for a set of heterogeneous disorders affecting kidney structures and characterized by a decline in renal function. Age-related renal insufficiency has important implications with regard to body homeostasis, drug toxicity and renal transplantation. In our study, two-photon microscopy was used to image kidney morphological and functional characteristics in an age-related rat model in vivo. The changes in morphology are analyzed based on autofluorescence and Hoechst 33342 labeling in rats with different ages. Structural parameters including renal tubular diameter, cell nuclei density, size and shape are studied and compared with Hematoxylin and Eosin histological analysis. Functional characteristics, such as blood flow, and glomerular filtration rate are studied with high-molecular weight (MW) 500-kDa dextran-fluorescein and low-MW 10-kDa dextran-rhodamine. Results indicate that morphology changes significantly and functional characteristics deteriorate with age. These parameters are potential indicators for evaluating age-related renal morphology and function changes. Combined analyses of these parameters could provide a quantitative, novel method for monitoring kidney diseases and/or therapeutic effects of kidney drugs.

Optical coherence tomography and computer-aided diagnosis of a murine model of chronic kidney disease.

Chronic kidney disease (CKD) is characterized by a progressive loss of renal function over time. Histopathological analysis of the condition of glomeruli and the proximal convolutional tubules over time can provide valuable insights into the progression of CKD. Optical coherence tomography (OCT) is a technology that can analyze the microscopic structures of a kidney in a nondestructive manner. Recently, we have shown that OCT can provide real-time imaging of kidney microstructures in vivo without administering exogenous contrast agents. A murine model of CKD induced by intravenous Adriamycin (ADR) injection is evaluated by OCT. OCT images of the rat kidneys have been captured every week up to eight weeks. Tubular diameter and hypertrophic tubule population of the kidneys at multiple time points after ADR injection have been evaluated through a fully automated computer-vision system. Results revealed that mean tubular diameter and hypertrophic tubule population increase with time in post-ADR injection period. The results suggest that OCT images of the kidney contain abundant information about kidney histopathology. Fully automated computer-aided diagnosis based on OCT has the potential for clinical evaluation of CKD conditions.

The preparation, characterization, and application of environment-friendly monoclonal antibodies for human blood cell.

OBJECTIVES: Monoclonal anti-human blood group A (51A8) and B (63B6) antibody reagents were prepared using the serum-free technique. The aims of this research were to characterize the serum-free reagents and prove their reliabilities in routine use. METHODS: Experiments including antigen-antibody agglutination testing, stability testing, SDS-PAGE, protein and IgM quantification, flow cytometry, and variable domain sequencing were performed to characterize the anti-A (51A8) and anti-B (63B6) reagents. Over 12 000 samples were tested using these reagents as routine blood grouping reagents. RESULTS: Serum-free anti-A (51A8) and anti-B (63B6) reagents were stable in longitudinal and accelerated testing, and their high purity was shown in SDS-PAGE and IgM quantification. These reagents have high specificity to red blood cells in serologic agglutination testing and flow cytometric analysis. A1 and A2 subgroup antigens can be distinguished clearly by patterns of flow cytometric histograms. No discrepancy was found in clinical trials of 12 000 samples. DISCUSSION: To reduce the risk of being affected by any animal additives, a serum-free culture system was applied to get mass-production of monoclonal anti-A/B antibodies. The high specificity and the high purity of the reagents were verified by the lab experiments. CONCLUSION: Lab research and clinical trial showed that serum-free monoclonal anti-A (51A8) and anti-B (63B6) reagents meet the requirements of routine blood grouping reagents. Moreover, these reagents featured ultra-high purity that is missing in other commercial counterparts, and therefore are recommended as more environment-friendly reagents.

Multi-modality Optical Imaging of Rat Kidney Dysfunction: In Vivo Response to Various Ischemia Times.

We observed in vivo kidney dysfunction with various ischemia times at 30, 75, 90, and 120 min using multi-modality optical imaging: optical coherence tomography (OCT), Doppler OCT (DOCT), and two-photon microscopy (TPM). We imaged the renal tubule lumens and glomerulus at several areas of each kidney before, during, and after ischemia of 5-month-old female Munich-Wistar rats. For animals with 30 and 75 min ischemia times, we observed that all areas were recovered after ischemia, that tubule lumens were re-opened and the blood flow of the glomerulus was re-established. For animals with 90 and 120 min ischemia times, we observed unrecovered areas, and that tubule lumens remained close after ischemia. TPM imaging verified the results of OCT and provided higher resolution images than OCT to visualize renal tubule lumens and glomerulus blood flow at the cellular level.

Optical Coherence Tomography of the Aging Kidney.

OBJECTIVES: The aging kidney exhibits a progressive decline in renal function with characteristic histopathologic changes and is a risk factor for renal transplant. However, the degree to which the kidney exhibits this decline depends on several factors that vary from one individual to the next. Optical coherence tomography is an evolving noninvasive imaging technology that has recently been used to evaluate acute tubular necrosis of living-human donor kidneys before their transplant. With the increasing use of kidneys from older individuals, it is important to determine whether optical coherence tomography also can distinguish the histopathology associated with aging. MATERIALS AND METHODS: In this investigation, we used Munich-Wistar rats to evaluate the ability of optical coherence tomography to detect histopathologic changes associated with aging. Optical coherence tomography observations were correlated with renal function and conventional light microscopic evaluation of these same kidneys. RESULTS: With the onset of severe proteinuria at 10 to 12 months of age, optical coherence tomography revealed tubular necrosis/atrophy, interstitial fibrosis, tubular dilation, and glomerulosclerosis. With a further deterioration in kidney function at 16 to 18 months of age (as indicated by rising creatinine levels), optical coherence tomography revealed more extensive interstitial fibrosis and tubular atrophy, increased tubular dilation with cyst formation and more sclerotic glomeruli. CONCLUSIONS: The foregoing observations suggest that optical coherence tomography can be used to detect the histopathology of progressive nephropathy associated with aging.

Spatial, temporal, and quantitative manipulation of intracellular hydrogen peroxide in cultured cells.

Hydrogen peroxide (H2O2) is produced endogenously in a number of cellular compartments, including the mitochondria, the endoplasmic reticulum, peroxisomes, and at the plasma membrane, and can play divergent roles as a second messenger or a pathological toxin. It is assumed that the tuned production of H2O2 within neuronal and nonneuronal cells regulates a discreet balance between survival and death. However, a major challenge in understanding the physiological versus pathological role of H2O2 in cells has been the lack of validated methods that can spatially, temporally, and quantitatively modulate H2O2 production. A promising means of regulating endogenous H2O2 is through the expression of peroxide-producing enzyme d-amino acid oxidase (DAAO from Rhodotorula gracilis lacking a peroxisomal targeting sequence). Using viral vectors to express DAAO in distinct cell types and using targeting sequences to target DAAO to distinct subcellular sites, we can manipulate H2O2 production by applying the substrate d-alanine or permeable analogs of d-alanine. In this chapter, we describe the use of DAAO to produce H2O2 in culture models and the real-time visual validation of this technique using two-photon microscopy and chemoselective fluorescent probes.

Recent advances in hydrogen peroxide imaging for biological applications.

Mounting evidence supports the role of hydrogen peroxide (H2O2) in physiological signaling as well as pathological conditions. However, the subtleties of peroxide-mediated signaling are not well understood, in part because the generation, degradation, and diffusion of H2O2 are highly volatile within different cellular compartments. Therefore, the direct measurement of H2O2 in living specimens is critically important. Fluorescent probes that can detect small changes in H2O2 levels within relevant cellular compartments are important tools to study the spatial dynamics of H2O2. To achieve temporal resolution, the probes must also be photostable enough to allow multiple readings over time without loss of signal. Traditional fluorescent redox sensitive probes that have been commonly used for the detection of H2O2 tend to react with a wide variety of reactive oxygen species (ROS) and often suffer from photostablilty issues. Recently, new classes of H2O2 probes have been designed to detect H2O2 with high selectivity. Advances in H2O2 measurement have enabled biomedical scientists to study H2O2 biology at a level of precision previously unachievable. In addition, new imaging techniques such as two-photon microscopy (TPM) have been employed for H2O2 detection, which permit real-time measurements of H2O2 in vivo. This review focuses on recent advances in H2O2 probe development and optical imaging technologies that have been developed for biomedical applications.

B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS.

Activation of intrinsic growth programs that promote developmental axon growth may also facilitate axon regeneration in injured adult neurons. Here, we demonstrate that conditional activation of B-RAF kinase alone in mouse embryonic neurons is sufficient to drive the growth of long-range peripheral sensory axon projections in vivo in the absence of upstream neurotrophin signaling. We further show that activated B-RAF signaling enables robust regenerative growth of sensory axons into the spinal cord after a dorsal root crush as well as substantial axon regrowth in the crush-lesioned optic nerve. Finally, the combination of B-RAF gain-of-function and PTEN loss-of-function promotes optic nerve axon extension beyond what would be predicted for a simple additive effect. We conclude that cell-intrinsic RAF signaling is a crucial pathway promoting developmental and regenerative axon growth in the peripheral and central nervous systems.

Two-photon fluorescence imaging of intracellular hydrogen peroxide with chemoselective fluorescent probes.

We present the application of two-photon fluorescence (TPF) imaging to monitor intracellular hydrogen peroxide (H2O2) production in brain cells. For selective imaging of H2O2 over other reactive oxygen species, we employed small-molecule fluorescent probes that utilize a chemoselective boronate deprotection mechanism. Peroxyfluor-6 acetoxymethyl ester detects global cellular H2O2 and mitochondria peroxy yellow 1 detects mitochondrial H2O2. Two-photon absorption cross sections for these H2O2 probes are measured with a mode-locked Ti:sapphire laser in the wavelength range of 720 to 1040 nm. TPF imaging is demonstrated in the HT22 cell line to monitor both cytoplasmic H2O2 and localized H2O2 production in mitochondria. Endogenous cytoplasmic H2O2 production is detected with TPF imaging in rat astrocytes modified with d-amino acid oxidase. The TPF H2O2 imaging demonstrated that these chemoselective probes are powerful tools for the detection of intracellular H2O2.

CD36 is involved in astrocyte activation and astroglial scar formation.

Inflammation is an essential component for glial scar formation. However, the upstream mediator(s) that triggers the process has not been identified. Previously, we showed that the expression of CD36, an inflammatory mediator, occurs in a subset of astcotyes in the peri-infarct area where the glial scar forms. This study investigates a role for CD36 in astrocyte activation and glial scar formation in stroke. We observed that the expression of CD36 and glial fibrillary acidic protein (GFAP) coincided in control and injured astrocytes and in the brain. Furthermore, GFAP expression was attenuated in CD36 small interfering RNA transfected astrocytes or in the brain of CD36 knockout (KO) mice, suggesting its involvement in GFAP expression. Using an in-vitro model of wound healing, we found that CD36 deficiency attenuated the proliferation of astrocytes and delayed closure of the wound gap. Furthermore, stroke-induced GFAP expression and scar formation were significantly attenuated in the CD36 KO mice compared with wild type. These findings identify CD36 as a novel mediator for injury-induced astrogliosis and scar formation. Targeting CD36 may serve as a potential strategy to reduce glial scar formation in stroke.

Mithramycin is a gene-selective Sp1 inhibitor that identifies a biological intersection between cancer and neurodegeneration.

Oncogenic transformation of postmitotic neurons triggers cell death, but the identity of genes critical for degeneration remain unclear. The antitumor antibiotic mithramycin prolongs survival of mouse models of Huntington's disease in vivo and inhibits oxidative stress-induced death in cortical neurons in vitro. We had correlated protection by mithramycin with its ability to bind to GC-rich DNA and globally displace Sp1 family transcription factors. To understand how antitumor drugs prevent neurodegeneration, here we use structure-activity relationships of mithramycin analogs to discover that selective DNA-binding inhibition of the drug is necessary for its neuroprotective effect. We identify several genes (Myc, c-Src, Hif1α, and p21(waf1/cip1)) involved in neoplastic transformation, whose altered expression correlates with protective doses of mithramycin or its analogs. Most interestingly, inhibition of one these genes, Myc, is neuroprotective, whereas forced expression of Myc induces Rattus norvegicus neuronal cell death. These results support a model in which cancer cell transformation shares key genetic components with neurodegeneration.

Controlled enzymatic production of astrocytic hydrogen peroxide protects neurons from oxidative stress via an Nrf2-independent pathway.

Neurons rely on their metabolic coupling with astrocytes to combat oxidative stress. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) appears important for astrocyte-dependent neuroprotection from oxidative insults. Indeed, Nrf2 activators are effective in stroke, Parkinson disease, and Huntington disease models. However, key endogenous signals that initiate adaptive neuroprotective cascades in astrocytes, including activation of Nrf2-mediated gene expression, remain unclear. Hydrogen peroxide (H(2)O(2)) plays an important role in cell signaling and is an attractive candidate mediator of adaptive responses in astrocytes. Here we determine (i) the significance of H(2)O(2) in promoting astrocyte-dependent neuroprotection from oxidative stress, and (ii) the relevance of H(2)O(2) in inducing astrocytic Nrf2 activation. To control the duration and level of cytoplasmic H(2)O(2) production in astrocytes cocultured with neurons, we heterologously expressed the H(2)O(2)-producing enzyme Rhodotorula gracilis D-amino acid oxidase (rgDAAO) selectively in astrocytes. Exposure of rgDAAO-astrocytes to D-alanine lead to the concentration-dependent generation of H(2)O(2). Seven hours of low-level H(2)O(2) production (∼3.7 nmol·min·mg protein) in astrocytes protected neurons from oxidative stress, but higher levels (∼130 nmol·min·mg protein) were neurotoxic. Neuroprotection occurred without direct neuronal exposure to astrocyte-derived H(2)O(2), suggesting a mechanism specific to astrocytic intracellular signaling. Nrf2 activation mimicked the effect of astrocytic H(2)O(2) yet H(2)O(2)-induced protection was independent of Nrf2. Astrocytic protein tyrosine phosphatase inhibition also protected neurons from oxidative death, representing a plausible mechanism for H(2)O(2)-induced neuroprotection. These findings demonstrate the utility of rgDAAO for spatially and temporally controlling intracellular H(2)O(2) concentrations to uncover unique astrocyte-dependent neuroprotective mechanisms.

Two-photon bioimaging utilizing supercontinuum light generated by a high-peak-power picosecond semiconductor laser source.

We developed a novel scheme for two-photon fluorescence bioimaging. We generated supercontinuum (SC) light at wavelengths of 600 to 1200 nm with 774-nm light pulses from a compact turn-key semiconductor laser picosecond light pulse source that we developed. The supercontinuum light was sliced at around 1030- and 920-nm wavelengths and was amplified to kW-peak-power level using laboratory-made low-nonlinear-effects optical fiber amplifiers. We successfully demonstrated two-photon fluorescence bioimaging of mouse brain neurons containing green fluorescent protein (GFP).

Two-photon fluorescence bioimaging with an all-semiconductor laser picosecond pulse source.

We have demonstrated successful two-photon excitation fluorescence bioimaging using a high-power pulsed all-semiconductor laser. Toward this purpose, we developed a pulsed light source consisting of a mode-locked laser diode and a two-stage diode laser amplifier. This pulsed light source provided optical pulses of 5 ps duration and having a maximum peak power of over 100 W at a wavelength of 800 nm and a repetition frequency of 500 MHz.

Two-photon bioimaging with picosecond optical pulses from a semiconductor laser.

Toward a practical light source for two-photon bioimaging, we have generated kilowatt peak power of 0.77 mum wavelength and 5 ps optical pulse via second-harmonic generation of the amplified output from a gain-switched 1.55 mum semiconductor laser. This compact scheme and stable optical-pulse-source has been successfully used for the two-photon fluorescence bioimaging of actin filaments in PtK2 cells.

Fabrication of beam shapers in the bulk of fused silica by femtosecond laser pulses.

We reported a new approach to the fabrication of three-dimensional refractive-index-modified microstructures inside transparent materials by combining two-dimensional writing by scanning the focus of the femtosecond laser pulse and by forming the long filament in the third dimension. In this way, embedded diffractive beam shapers of grid, square, and ring gratings were obtained in the bulk of fused silica by use of a femtosecond laser with a wavelength of 810 nm and a repetition rate of 1 KHz. These structures and their refractive efficiencies were optimized by selection of the appropriate fabrication parameters, including the pulse energy, grating period, scanning speed, and scanning repetition. The good performance of these devices indicates that, owing to its simple and flexible method for fabricating complex phase elements inside transparent materials, this technique has potential applications to integrated optics.

Wavy lines in two-photon photopolymerization microfabrication.

We present experimental results on the changes of lines in two-photon photopolymerization microfabrication. Polymerized lines remain straight, become wavy, and even float away with increased focus height as the sample is moved closer to the focusing lens. The influence of the focus height, the incident laser energy, and the scan speed was studied. The lower the incident energy or the faster the scan speed, the more easily the lines become wavy. From the focus height at which the lines become wavy and float away, we can estimate the lateral and longitudinal size of a voxel.

Multiple foci and a long filament observed with focused femtosecond pulse propagation in fused silica.

Multiple foci and a long filament are observed when we focus a femtosecond laser pulse into a fused-silica sample. The dependences of the intensity distribution of the plasma luminescence on the pulse energy and the numerical aperture (NA) of the focusing objective are investigated. Multiple foci are observed when NA of