A Proteomic Approach for Comprehensively Screening Substrates of Protein Kinases Such as Rho-Kinase

BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity

Transmission of Lamb waves across a partially closed crack: Numerical analysis and experiment

The transmission characteristics of Lamb waves across a partially closed through-thickness crack in a plate are investigated numerically and experimentally. In the numerical analysis, the spectral element method is used to simulate the transmission of the lowest-order symmetric (S0) and antisymmetric (A0) Lamb modes across a crack in a low-frequency range. The analysis is carried out for an open crack with traction-free surfaces as well as for a partially closed crack modeled as a spring-type interface characterized by normal and tangential stiffnesses. The transmission ratios of both modes are obtained from the spectral amplitude of the simulated transmission waveforms for different crack lengths and interfacial stiffnesses. The numerical results show that the transmission ratio of the S0 mode increases monotonically with the interfacial stiffness, but that of the A0 mode depends on the interfacial stiffness in a non-monotonic manner depending on the frequency. The Lamb wave transmission measurements are carried out for aluminum alloy plates with artificial slits or a fatigue crack. The experimental results for the plates with slits show reasonable agreement with the numerical results for open cracks. The measured transmission ratio of the S0 mode is shown to decrease with the tensile load applied to the plate, but that of the A0 mode shows different load dependence for different frequencies. The qualitative features of the experimental results for the fatigue crack are discussed based on the numerical simulation for closed cracks

Dietary Iron Overload Differentially Modulates Chemically-Induced Liver Injury in Rats

Hepatic iron overload is well known as an important risk factor for progression of liver diseases; however, it is unknown whether it can alter the susceptibility to drug-induced hepatotoxicity. Here we investigate the pathological roles of iron overload in two single-dose models of chemically-induced liver injury. Rats were fed a high-iron (Fe) or standard diet (Cont) for four weeks and were then administered with allyl alcohol (AA) or carbon tetrachloride (CCl4). Twenty-four hours after administration mild mononuclear cell infiltration was seen in the periportal/portal area (Zone 1) in Cont-AA group, whereas extensive hepatocellular necrosis was seen in Fe-AA group. Centrilobular (Zone 3) hepatocellular necrosis was prominent in Cont-CCl4 group, which was attenuated in Fe-CCl4 group. Hepatic lipid peroxidation and hepatocellular DNA damage increased in Fe-AA group compared with Cont-AA group. Hepatic caspase-3 cleavage increased in Cont-CCl4 group, which was suppressed in Fe-CCl4 group. Our results showed that dietary iron overload exacerbates AA-induced Zone-1 liver injury via enhanced oxidative stress while it attenuates CCl4-induced Zone-3 liver injury, partly via the suppression of apoptosis pathway. This study suggested that susceptibility to drugs or chemical compounds can be differentially altered in iron-overloaded livers

Single-Cell Memory Regulates a Neural Circuit for Sensory Behavior

Unveiling the molecular and cellular mechanisms underlying memory has been a challenge for the past few decades. Although synaptic plasticity is proven to be essential for memory formation, the significance of “single-cell memory” still remains elusive. Here, we exploited a primary culture system for the analysis of C. elegans neurons and show that a single thermosensory neuron has an ability to form, retain, and reset a temperature memory. Genetic and proteomic analyses found that the expression of the single-cell memory exhibits inter-individual variability, which is controlled by the evolutionarily conserved CaMKI/IV and Raf pathway. The variable responses of a sensory neuron influenced the neural activity of downstream interneurons, suggesting that modulation of the sensory neurons ultimately determines the behavioral output in C. elegans. Our results provide proof of single-cell memory and suggest that the individual differences in neural responses at the single-cell level can confer individuality

The draft genome sequence of the Japanese rhinoceros beetle Trypoxylus dichotomus septentrionalis towards an understanding of horn formation

: The Japanese rhinoceros beetle Trypoxylus dichotomus is a giant beetle with distinctive exaggerated horns present on the head and prothoracic regions of the male. T. dichotomus has been used as a research model in various fields such as evolutionary developmental biology, ecology, ethology, biomimetics, and drug discovery. In this study, de novo assembly of 615 Mb, representing 80% of the genome estimated by flow cytometry, was obtained using the 10 × Chromium platform. The scaffold N50 length of the genome assembly was 8.02 Mb, with repetitive elements predicted to comprise 49.5% of the assembly. In total, 23,987 protein-coding genes were predicted in the genome. In addition, de novo assembly of the mitochondrial genome yielded a contig of 20,217 bp. We also analyzed the transcriptome by generating 16 RNA-seq libraries from a variety of tissues of both sexes and developmental stages, which allowed us to identify 13 co-expressed gene modules. We focused on the genes related to horn formation and obtained new insights into the evolution of the gene repertoire and sexual dimorphism as exemplified by the sex-specific splicing pattern of the doublesex gene. This genomic information will be an excellent resource for further functional and evolutionary analyses, including the evolutionary origin and genetic regulation of beetle horns and the molecular mechanisms underlying sexual dimorphism

Detection of Low-Signal Pulvinar Areas Using Diffusion-Weighted Imaging in Patients with Dementia Experiencing Visual Hallucinations

Background: Little research has been conducted regarding the role of pulvinar nuclei in the pathogenesis of visual hallucinations due to the difficulty of assessing abnormalities in this region using conventional magnetic resonance imaging (MRI). The present study aimed to retrospectively investigate the relative abilities of diffusion-weighted imaging (DWI), fluid-attenuated inversion recovery (FLAIR), and susceptibility-weighted imaging (SWI) to visualize the pulvinar and to ascertain the relationship between pulvinar visualization and visual hallucinations. Methods: A retrospective analysis of 3T MRIs from 73 patients (31 males, 42 females; mean age 73.5 ± 12.7 years) of the Memory Clinic of Mie University Hospital was conducted. Correlations between pulvinar visualization and the following were analyzed: age, sex, education, hypertension, hyperlipidemia, diabetes mellitus, Mini-Mental State Examination score, Evans index, and visual hallucinations. Results: DWI detected low-signal pulvinar areas in approximately half of the patients (52.1%). Participants with pulvinar visualization were significantly older, and the pulvinar was more frequently visualized in patients who had experienced visual hallucinations compared to those who had not. No significant association was observed between whole brain atrophy and pulvinar visualization. Conclusions: The results of the present study indicate that diffusion-weighted 3T MRI is the most suitable method for the detection of pulvinar nuclei in patients with dementia experiencing visual hallucinations

Single-Cell Memory Regulates a Neural Circuit for Sensory Behavior

Unveiling the molecular and cellular mechanisms underlying memory has been a challenge for the past few decades. Although synaptic plasticity is proven to be essential for memory formation, the significance of "single-cell memory" still remains elusive. Here, we exploited a primary culture system for the analysis of C. elegans neurons and show that a single thermosensory neuron has an ability to form, retain, and reset a temperature memory. Genetic and proteomic analyses found that the expression of the single- cell memory exhibits inter-individual variability, which is controlled by the evolutionarily conserved CaMKI/IV and Raf pathway. The variable responses of a sensory neuron influenced the neural activity of downstream interneurons, suggesting that modulation of the sensory neurons ultimately determines the behavioral output in C. elegans. Our results provide proof of single-cell memory and suggest that the individual differences in neural responses at the single-cell level can confer individuality.A single thermosensory neuron is capable of memorizing a temperature / This single-cell memory shows variability between individuals / The CaMKI/IV and Raf pathway modulates variability in single-cell memory / The variability of the single-cell memory underlies premotor neuron activit