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SUMMARY: DNA rearrangements, including inversions, translocations, and large insertions/deletions (indels), are crucial for crop evolution, domestication, and improvement. The rearrangements are frequently induced by ion beams via the mis‐repair of DNA double‐strand breaks (DSBs). Unfortunately, how ion beam‐induced DSBs are repaired has not been comprehensively analyzed and the mechanisms underlying DNA rearrangements remain unclear. In this study, clonal sectors originating from single mutated cells in carbon ion‐irradiated plants were used for whole‐genome sequencing analyses after Arabidopsis seeds and seedlings were irradiated. Comparative analyses of the induced mutations (e.g., size and frequency of indels and microhomology at the junctions of the rearrangements) in the irradiated materials suggested that the broken/rejoined DSB ends were more extensively processed in seedlings than in seeds. A mutation to canonical non‐homologous end‐joining (c‐NHEJ), which is a DSB repair pathway with minimal processing of DSB ends, increased the sensitivity to ion beams more in the seeds than in the seedlings, which was consistent with the junction analysis results, indicative of the minor contribution of c‐NHEJ to the carbon ion‐induced DSB repair in seedlings. Considering the characteristics of the large templated insertions in irradiated seedlings, ion‐beam‐induced DSBs in seedlings are likely repaired primarily by a polymerase theta‐mediated pathway. Polymerase theta‐deficient seedlings were more sensitive to ion beams than the c‐NHEJ‐deficient seedlings, consistent with this hypothesis. This study revealed the key characteristics of ion beam‐induced DSBs and the associated repair mechanisms related to the physiological status of the irradiated materials, with implications for elucidating the occurrence and induction of rearrangements. Significance Statement: DNA rearrangements, which are critical for crop improvement, can be randomly induced in genomes by ion beams. Our genomic analysis of irradiated plants revealed significant differences in the characteristics of the DNA rearrangements in seeds and seedlings, providing important insights into double‐strand break repair mechanisms and the associated rearrangement formation related to the physiological status of the irradiated materials. [ABSTRACT FROM AUTHOR]

Human prion diseases (HPDs) are progressive and fatal neurodegenerative diseases caused by abnormal prion protein (PrPSc). They can be sporadic, genetic or acquired. Sporadic HPDs include sporadic Creutzfeldt–Jakob disease (CJD) and sporadic fatal insomnia. Genetic HPDs include genetic CJD, Gerstman–Sträussler–Scheinker disease and fatal familial insomnia. Acquired HPDs include Kuru, variant CJD and iatrogenic CJD. The World Health Organization clinical diagnostic criteria for HPDs include clinical findings, cerebrospinal fluid protein markers and electroencephalography, and the UK and European clinical diagnostic criteria include a combination of clinical findings, 14‐3‐3 protein in the cerebrospinal fluid, magnetic resonance imaging diffusion‐weighted imaging and electroencephalography. Recently, the development and clinical application of real‐time quaking‐induced conversion has allowed pre‐mortem diagnosis of sporadic CJD. The real‐time quaking‐induced conversion assay allows detection of >1 fg of PrPSc in diluted CJD brain homogenate, as well as a variety of biological tissues and cerebrospinal fluid. However, the real‐time quaking‐induced conversion assay does not provide prognostic data and has lower diagnostic accuracy for some rarer, atypical prion diseases. Furthermore, recent advancements in laboratory testing and magnetic resonance imaging diffusion‐weighted imaging have shown improved diagnostic accuracy for prion diseases. Clinical trials of prion disease treatments have recently begun in many countries. The therapeutic window consists mainly of the early stage of disease, and few clinical studies have examined the role of biomarkers at this stage. Therefore, biomarkers, imaging and clinical symptoms might be extremely important for the diagnosis of prion diseases. Clinical trials of prion disease treatments have recently begun in many countries. The therapeutic window mainly consists of the early stage of disease, and few clinical studies have examined the role of biomarkers at this stage. Therefore, biomarkers, imaging and clinical symptoms might be extremely important for the diagnosis of prion diseases. [ABSTRACT FROM AUTHOR]

Radiation-induced mutations have been detected by whole-genome sequencing analyses of self-pollinated generations of mutagenized plants. However, large DNA alterations and mutations in non-germline cells were likely missed. In this study, in order to detect various types of mutations in mutagenized M1 plants, anthocyanin pigmentation was used as a visible marker of mutations. Arabidopsis seeds heterozygous for the anthocyanin biosynthetic genes were irradiated with gamma-rays. Anthocyanin-less vegetative sectors resulting from a loss of heterozygosity were isolated from the gamma-irradiated M1 plants. The whole-genome sequencing analysis of the sectors detected various mutations, including structural variations (SVs) and large deletions (≥100 bp), both of which have been less characterized in the previous researches using gamma-irradiated plant genomes of M2 or later generations. Various types of rejoined sites were found in SVs, including no-insertion/deletion (indel) sites, only-deletion sites, only-insertion sites, and indel sites, but the rejoined sites with 0–5 bp indels represented most of the SVs. Examinations of the junctions of rearrangements (SVs and large deletions), medium deletions (10–99 bp), and small deletions (2–9 bp) revealed unique features (i.e., frequency of insertions and microhomology) at the rejoined sites. These results suggest that they were formed preferentially via different processes. Additionally, mutations that occurred in putative single M1 cells were identified according to the distribution of their allele frequency. The estimated mutation frequencies and spectra of the M1 cells were similar to those of previously analyzed M2 cells, with the exception of the greater proportion of rearrangements in the M1 cells. These findings suggest there are no major differences in the small mutations (<100 bp) between vegetative and germline cells. Thus, this study generated valuable information that may help clarify the nature of gamma-irradiation-induced mutations and their occurrence in cells that develop into vegetative or reproductive tissues. Author summary: Mutations that occur in plant genome are not only related to plant evolution and speciation in nature, and also useful to identify novel gene functions and to develop new cultivars. Ionizing radiations induce various types of mutations throughout genomes in individual cells of an irradiated/mutagenized plant. However, current knowledge on radiation-induced genome-wide mutations in plants relied on the analyses of self-pollinated generations (M2 or later generations) of the mutagenized plants (M1 generation). Thus, mutations that are hardly transmitted to the next generation and those occurred in non-germline cells could not be investigated. Here, using anthocyanin pigmentation as a visible marker to reduce the genomic complexity in M1 plants, we achieved reliable detection of radiation-induced genome-wide mutations. We demonstrated that rearrangements, which were hardly detected in previous analyses using M2 genomes, occurred substantially often in gamma-irradiated M1 cells. We also revealed that mutation profile of the M1 cells was comparable with that of M2 genomes reported in previous analyses, except for the higher proportion of rearrangements in the M1 genome. Together with unique features at rejoined sites of rearrangements, medium deletions, and small deletions in the M1 genome, our findings are helpful to know the nature of genome-wide mutations induced by gamma-irradiation. [ABSTRACT FROM AUTHOR]

Cellular prion protein (PrP) is a membrane protein that is highly conserved among mammals and mainly expressed on the cell surface of neurons. Despite its reported interactions with various membrane proteins, no functional studies have so far been carried out on it, and its physiological functions remain unclear. Neuronal cell death has been observed in a PrP-knockout mouse model expressing Doppel protein, suggesting that PrP might be involved in Ca2+ signaling. In this study, we evaluated the binding of PrP to metabotropic glutamate receptor 1 (mGluR1) and found that wild-type PrP (PrP-wt) and mGluR1 co-immunoprecipitated in dual-transfected Neuro-2a (N2a) cells. Fluorescence resonance energy transfer analysis revealed an energy transfer between mGluR1-Cerulean and PrP-Venus. In order to determine whether PrP can modulate mGluR1 signaling, we performed Ca2+ imaging analyses following repetitive exposure to an mGluR1 agonist. Agonist stimulation induced synchronized Ca2+ oscillations in cells coexpressing PrP-wt and mGluR1. In contrast, N2a cells expressing PrP-ΔN failed to show ligand-dependent regulation of mGluR1-Ca2+ signaling, indicating that PrP can bind to mGluR1 and modulate its function to prevent irregular Ca2+ signaling and that its N-terminal region functions as a molecular switch during Ca2+ signaling. • Prion protein (PrP) binds to metabotropic glutamate receptor 1 (mGluR1). • N-terminal region of PrP is essential for binding of PrP and mGluR1. • PrP modulates mGluR1 function to prevent irregular Ca2+ signaling. • N-terminal region of PrP functions as a molecular switch during Ca2+ signaling. [ABSTRACT FROM AUTHOR]

Background: Recently, the investigation of cerebrospinal fluid (CSF) biomarkers for diagnosing human prion diseases (HPD) has garnered significant attention. Reproducibility and accuracy are paramount in biomarker research, particularly in the measurement of total tau (T-tau) protein, which is a crucial diagnostic marker. Given the global impact of the coronavirus disease pandemic, the frequency of measuring this protein using one of the world's fully automated assays, chemiluminescent enzyme immunoassay (CLEA), has increased. At present, the diagnosis and monitoring of neurological diseases mainly rely on traditional methods, but their accuracy and responsiveness are limited. There is limited knowledge of the accuracy of CLEA in tau measurements. We aimed to measure T-tau protein using CLEA and to elucidate its merits and limitations. Methods: We randomly selected 60 patients with rapidly progressive dementia, using ELISA and CLEA analysis of cerebrospinal fluid specimens. Additionally, we used Western blotting to detect the presence of 14-3-3 protein and employed real-time quaking-induced conversion (RT-QuIC) assays to analyze the same set of samples. Furthermore, we examined the correlation coefficient between ELISA and CLEA results in a subset of 60 samples. Moreover, using CLEA, we evaluated the diurnal reproducibility, storage stability, dilutability, and freeze–thaw effects in three selected samples. Results: In 172 patients, 172 samples were extracted, with each patient providing only one sample, and a total of 88 (35 men and 53 women) tested positive for HPD in the RT-QuIC assay. In contrast, all CSF samples from the remaining 84 patients without HPD (50 men and 34 women) tested negative in the RT-QuIC assay. Both ELISA and CLEA showed perfect sensitivity and specificity (100%) in measuring T-tau protein levels. In addition, ELISA and CLEA are similar in terms of measurement sensitivity and marginal effect of detection extrema. CLEA analysis exhibited instability for certain samples with T-tau protein levels exceeding 2000 pg/mL, leading to low reproducibility during dilution analysis. Conclusions: Our findings indicate that CLEA outperforms ELISA in terms of diurnal reproducibility, storage stability, and freeze–thaw effects. However, ELISA demonstrated superior performance in the dilution assay. Therefore, it is imperative to develop innovative approaches for the dilution of biomarker samples for CLEA measurements during clinical trials. [ABSTRACT FROM AUTHOR]

New, useful microorganism resources have been generated by ionizing radiation breeding technology. However, the mutagenic effects of ionizing radiation on microorganisms have not been systematically clarified. For a deeper understanding and characterization of ionizing radiation-induced mutations in microorganisms, we investigated the lethal effects of seven different linear energy transfer (LET) radiations based on the survival fraction (SF) and whole-genome sequencing analysis of the mutagenic effects of a dose resulting in an SF of around 1% in Bacillus subtilis spores. Consequently, the lower LET radiations (gamma [surface LET: 0.2 keV/µm] and 4He2+ [24 keV/µm]) showed low lethality and high mutation frequency (MF), resulting in the major induction of single-base substitutions. Whereas higher LET radiations (12C5+ [156 keV/µm] and 12C6+ [179 keV/µm]) showed high lethality and low MF, resulting in the preferential induction of deletion mutations. In addition, 12C6+ (111) ion beams likely possess characteristics of both low- and high-LET radiations simultaneously. A decrease in the relative biological effectiveness and an evaluation of the inactivation cross section indicated that 20Ne8+ (468 keV/µm) and 40Ar13+ (2214 keV/µm) ion beams had overkill effects. In conclusion, in the mutation breeding of microorganisms, it should be possible to regulate the proportions, types, and frequencies of induced mutations by selecting an ionizing radiation of an appropriate LET in accordance with the intended purpose. • The proportions of mutation types varied with LET of radiation. • Ionizing radiations induce various SBS mutations without a large bias. • High-LET radiations induce a relatively high proportion of high-impact mutations. • Ionizing radiation may be a trigger of large-scale inversions. [ABSTRACT FROM AUTHOR]

Objective We investigated the relationships between the efficiency of intercellular transmission of human T-lymphotropic virus type I ( HTLV-I) and C-X-C chemokine receptor type 4 ( CXCR4) signaling in a comparative study of HTLV-I-infected T cell lines derived from an HTLV-I-associated myelopathy/tropical spastic paraparesis patient ( HCT-5) or an HTLV-I carrier ( TL-Su). Methods After co-cultivation of each HTLV-I-infected T cell line with H9/K30 luc reporter cells, relative activities of luc were calculated to measure the intercellular transmission of HTLV-I. The level of stromal cell-derived factor-1α was measured using an enzyme-linked immunoassay. Rap1 activation in HTLV-I-infected T cell lines was analyzed by a pull-down assay. Results HCT-5 had higher intercellular transmission efficiency of HTLV-I compared with TL-Su. Intercellular transmission of HTLV-I in TL-Su was increased significantly by stromal cell-derived factor-1α treatment. Pretreatment with an anti- CXCR4 blocking antibody for H9/K30 luc reporter cells induced a significant reduction in intercellular transmission of HTLV-I in HCT-5, but not in TL-Su. Stromal cell-derived factor-1α was not detected in the culture supernatants of HCT-5, but existence of a humoral factor that activates CXCR4 signaling in HCT-5 was suggested from studies with condition media. Bound Rap1- GTP was detected in HCT-5, but not in TL-Su. Conclusions Our data suggest that CXCR4 signaling is involved in the efficiency of intercellular transmission of HTLV-I. [ABSTRACT FROM AUTHOR]

The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrPSc. Real-time quaking-induced conversion can amplify very small amounts of PrPSc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt–Jakob disease patients demonstrated that 50% seeding dose (SD50) is reached approximately 1010/g brain (values varies 108.79–10.63/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrPSc concentrations in the samples, determined by dot-blot assay, was 0.6–5.4 μg/g brain; therefore, we estimated that 1 SD50 unit was equivalent to 0.06–0.27 fg of PrPSc. The SD50 values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission. [ABSTRACT FROM AUTHOR]

Entomopathogenic fungi, such asMetarhizium anisopliae(Metch.) Sorokin, are important agents for the biological control of insect pests. However, these fungi are not compatible with high temperatures. In this study, mutagenesis using ion beams or gamma rays was used to generate five potentially thermotolerant mutants from two wild-type isolates ofM. anisopliae(two using ion beams and three using gamma rays). The mutant isolates had a higher upper thermal limit for vegetative growth compared to the wild types (by 2–3°C) and enhanced tolerance to wet–heat stress of 45°C for conidial germination. At 25°C and 30°C, most mutants were as virulent to maize weevil adults as the wild type, however, one mutant produced using ion beams almost lost virulence entirely. These results indicate that ion beams and gamma rays are useful tools for improving biological characteristics, such as thermotolerance, in entomopathogenic fungi, but that mutants must be carefully evaluated for unpredictable negative side effects. [ABSTRACT FROM AUTHOR]

The main therapeutic strategy against human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) characterized by lower extremity motor dysfunction is immunomodulatory treatment, with drugs such as corticosteroid hormone and interferon-α, at present. However, there are many issues in long-term treatment with these drugs, such as insufficient effects and various side effects. We now urgently need to develop other therapeutic strategies. The heparinoid, pentosan polysulfate sodium (PPS), has been safely used in Europe for the past 50 years as a thrombosis prophylaxis and for the treatment of phlebitis. We conducted a clinical trial to test the effect of subcutaneous administration of PPS in 12 patients with HAM/TSP in an open-labeled design. There was a marked improvement in lower extremity motor function, based on reduced spasticity, such as a reduced time required for walking 10 m and descending a flight of stairs. There were no significant changes in HTLV-I proviral copy numbers in peripheral blood contrary to the inhibitory effect of PPS in vitro for intercellular spread of HTLV-I. However, serum soluble vascular cell adhesion molecule (sVCAM)-1 was significantly increased without significant changes of serum level of chemokines (CXCL10 and CCL2). There was a positive correlation between increased sVCAM-1and reduced time required for walking 10 m. PPS might induce neurological improvement by inhibition of chronic inflammation in the spinal cord, through blocking the adhesion cascade by increasing serum sVCAM-1, in addition to rheological improvement of the microcirculation. PPS has the potential to be a new therapeutic tool for HAM/TSP. [ABSTRACT FROM AUTHOR]

Objective To investigate the relationship between the intercellular transmission efficiency of human T-lymphotropic virus type I ( HTLV-I) and the signaling involved in actin polymerization during cytoskeletal reorganization in a comparative study of HTLV-I-infected T-cell lines derived from an HTLV-I-associated myelopathy/tropical spastic paraparesis ( HAM/ TSP) patient or an HTLV-I carrier. Methods HCT-5 and TL-Su cells derived from an HAM/TSP patient and an HTLV-I carrier, respectively, were used as HTLV-I-infected T-cell lines. After co-cultivation of each HTLV-I-infected T-cell line with H9/K30 luc reporter cells, the relative luc activities were calculated to analyze the efficiency of intercellular transmission of HTLV-I. The intracellular levels of cyclic adenosine monophosphate (c AMP) or cyclic guanosine monophosphate (c GMP) were measured in enzyme-linked immunoassays. The expression of phosphorylated vasodilator-stimulated phosphoprotein (p-VASP) was analyzed by western blotting. Results Treatment of HCT-5 cells with latrunculin B, an inhibitor of actin polymerization, significantly suppressed the relative luc activity. Western blotting analysis of HCT-5 cells treated with the adenylyl cyclase activator forskolin showed upregulation of p-VASP, with a concomitant and significant increase in the intracellular c AMP concentration. Furthermore, the relative luc activity was significantly decreased. The intracellular c AMP, but not c GMP levels, were significantly lower in HCT-5 than in TL-Su. Vasodilator-stimulated phosphoprotein appeared less phosphorylated in HCT-5 than in TL-Su. The relative luc activity was significantly higher in HCT-5 than in TL-Su. Conclusions The intracellular c AMP concentration regulates the efficiency of intercellular HTLV-I transmission under the control of p-VASP expression, suggesting the intercellular transmission potential of HTLV-I-infected T cells of HAM/TSP patients is enhanced by downregulated intracellular c AMP levels. [ABSTRACT FROM AUTHOR]

Recently, clinical trials of human prion disease (HPD) treatments have begun in many countries, and the therapeutic window of these trials focuses mainly on the early stage of the disease. Furthermore, few studies have examined the role of biomarkers at the early stage. According to the World Health Organization, the clinical diagnostic criteria for HPDs include clinical findings, cerebrospinal fluid (CSF) protein markers, and electroencephalography (EEG). In contrast, the UK and European clinical diagnostic criteria include a combination of clinical findings, 14-3-3 protein in the CSF, magnetic resonance imaging-diffusion-weighted imaging (MRI-DWI), and EEG. Moreover, recent advancements in laboratory testing and MRI-DWI have improved the accuracy of diagnostics used for prion diseases. However, according to MRI-DWI data, patients with rapidly progressing dementia are sometimes misdiagnosed with HPD due to the high-intensity areas detected in their brains. Thus, analyzing the CSF biomarkers is critical to diagnose accurately different diseases. CSF biomarkers are investigated using a biochemical approach or the protein amplification methods that utilize the unique properties of prion proteins and the ability of PrPSc to induce a conformational change. The biochemical markers include the 14-3-3 and total tau proteins of the CSF. In contrast, the protein amplification methods include the protein misfolding cyclic amplification assay and real-time quaking-induced conversion (RT-QuIC) assay. The RT-QuIC analysis of the CSF has been proved to be a highly sensitive and specific test for identifying sporadic HPD forms; for this reason, it was included in the diagnostic criteria. • Real-time quaking-induced conversion (RT-QuIC) is a protein misfolding assay. • For CSF, this assay shows high sensitivity in identifying sporadic prion disease. • This assay for CSF also shows high specificity. [ABSTRACT FROM AUTHOR]

Orthologs of Escherichia coli ygjD and yeaZ genes are highly conserved in various organisms. The genome of the radioresistant bacterium Deinococcus radiodurans possesses single orthologs of ygjD (DR_0382) and yeaZ (DR_0756). Complete loss of either one or both genes did not result in any significant changes in cell growth efficiency, indicating that both genes are not essential for cell viability in D. radiodurans, unlike the case with other species such as E. coli, Bacillus subtilis and Saccharomyces cerevisiae. Survival rates following DNA damage induced by hydrogen peroxide (HO), N-methyl- N′-nitro- N-nitrosoguanidine (MNNG), ultra violet (UV) radiation, γ-rays, cisplatin and mitomycin C (MMC) were compared among the wild-type strain and D. radiodurans ygjD/ yeaZ null mutants. Cell viability of the null mutants did not decrease following exposure to HO or MNNG. In addition, the reduction in cell viability following exposure to γ-rays, UV radiation or cisplatin was marginal in the null mutants compared to the wild-type strain. Interestingly, the null mutants exhibited high sensitivity to MMC, which mainly causes interstrand DNA cross-links. The sensitivity of the null mutants to MMC was restored to that of the wild type by transformation with plasmids expressing these genes. These results suggest that D. radiodurans ygjD and yeaZ genes are involved in DNA repair and play a role in the repair of DNA cross-links. [ABSTRACT FROM AUTHOR]

Introduction: The definitive diagnosis of genetic prion diseases (gPrD) requires pathological confirmation. To date, diagnosis has relied upon the finding of the biomarkers 14-3-3 protein and total tau (t-tau) protein in the cerebrospinal fluid (CSF), but many researchers have reported that these markers are not sufficiently elevated in gPrD, especially in Gerstmann- Strãussler-Scheinker syndrome (GSS). We recently developed a new in vitro amplification technology, designated "real-time quaking-induced conversion (RT-QUIC)", to detect the abnormal form of prion protein in CSF from sporadic Creutzfeldt- Jakob disease (sCJD) patients. In the present study, we aimed to investigate the presence of biomarkers and evaluate RT- QUIC assay in patients with gPrD, as the utility of RT-QUIC as a diagnostic tool in gPrD has yet to be determined. Method/Principal Findings: 56 CSF samples were obtained from gPrD patients, including 20 cases of GSS with P102L mutation, 12 cases of fatal familial insomnia (FFI; D178N), and 24 cases of genetic CJD (gCJD), comprising 22 cases with E200K mutation and 2 with V203I mutation. We subjected all CSF samples to RT-QUIC assay, analyzed 14-3-3 protein by Western blotting, and measured t-tau protein using an ELISA kit. The detection sensitivities of RT-QUIC were as follows: GSS (78%), FFI (100%), gCJD E200K (87%), and gCJD V203I (100%). On the other hand the detection sensitivities of biomarkers were considerably lower: GSS (11%), FFI (0%), gCJD E200K (73%), and gCJD V203I (67%). Thus, RT-QUIC had a much higher detection sensitivity compared with testing for biomarkers, especially in patients with GSS and FFI. Conclusion/Significance: RT-QUIC assay is more sensitive than testing for biomarkers in gPrD patients. RT-QUIC method would thus be useful as a diagnostic tool when the patient or the patient's family does not agree to genetic testing, or to confirm the diagnosis in the presence of a positive result for genetic testing. [ABSTRACT FROM AUTHOR]

Abstract: Deinococcus radiodurans exhibits extraordinary resistance to the lethal effect of DNA-damaging agents, a characteristic attributed to its highly proficient DNA repair capacity. Although the D. radiodurans genome is clearly devoid of recBC and addAB counterparts as RecA mediators, the genome possesses all genes associated with the RecFOR pathway. In an effort to gain insights into the role of D. radiodurans RecFOR proteins in homologous recombination, we generated recF, recO and recR disruptant strains and characterized the disruption effects. All the disruptant strains exhibited delayed growth relative to the wild-type, indicating that the RecF, RecO and RecR proteins play an important role in cell growth under normal growth conditions. A slight reduction in transformation efficiency was observed in the recF and recO disruptant strains compared to the wild-type strain. Interestingly, disruption of recR resulted in severe reduction of the transformation efficiency. On the other hand, the recF disruptant strain was the most sensitive phenotype to γ rays, UV irradiation and mitomycin C among the three disruptants. In the recF disruptant strain, the intracellular level of the LexA1 protein did not decrease following γ irradiation, suggesting that a large amount of the RecA protein remains inactive despite being induced. These results demonstrate that the RecF protein plays a crucial role in the homologous recombination repair process by facilitating RecA activation in D. radiodurans. Thus, the RecF and RecR proteins are involved in the RecA activation and the stability of incoming DNA, respectively, during RecA-mediated homologous recombination processes that initiated the ESDSA pathway in D. radiodurans. Possible mechanisms that involve the RecFOR complex in homologous intermolecular recombination and homologous recombination repair processes are also discussed. [Copyright &y& Elsevier]

The development of technologies for the in vitro amplification of abnormal conformations of prion protein (PrPSc) has generated the potential for sensitive detection of prions. Here we developed a new PrPSc amplification assay, called real-time quaking-induced conversion (RT-QUIC), which allows the detection of ≥1 fg of PrPSc in diluted Creutzfeldt-Jakob disease (CJD) brain homogenate. Moreover, we assessed the technique first in a series of Japanese subjects and then in a blind study of 30 cerebrospinal fluid specimens from Australia, which achieved greater than 80% sensitivity and 100% specificity. These findings indicate the promising enhanced diagnostic capacity of RT-QUIC in the antemortem evaluation of suspected CJD. [ABSTRACT FROM AUTHOR]

Creutzfeldt-Jakob disease (CJD) is a transmissible, fatal, neurodegenerative disease in humans. Recently, various drugs have been reported to be useful in the treatment of CJD; however, for such treatments to be useful it is essential to rapidly and accurately diagnose CJD. 124 CJD patients and 87 with other diseases causing rapid progressive dementia were examined. Cerebral spinal fluid (CSF) from CJD patients was analyzed by 2D-PAGE and the protein expression pattern was compared with that from healthy subjects. One of three CJD-specific spots was found to be fatty acid binding protein (FABP), and heart-type FABP (H-FABP) was analyzed as a new biochemical marker for CJD. H-FABP ELISA results were compared between CJD patients and patients with other diseases ( n = 211). Visual readout accuracy of the Rapicheck H-FABP test panel for CSF was analyzed using an independent measure of CSF H-FABP concentration. The distribution of H-FABP in the brains of CJD patients was examined by immunohistochemistry. ELISA sensitivity and specificity were 90.3% and 92.9%, respectively, and Rapicheck H-FABP sensitivity and specificity were 87.9% and 96.0%, respectively. ELISA and Rapicheck H-FABP assays provided comparable results for 14-3-3 protein and total tau protein. Elevated H-FABP levels were associated with an accumulation of abnormal prion protein, astrocytic gliosis, and neuronal loss in the cerebral cortices of CJD patients. In conclusion, Rapicheck H-FABP of CSF specimens enabled quick and frequent diagnosis of CJD. H-FABP represents a new biomarker for CJD distinct from 14-3-3 protein and total tau protein. [ABSTRACT FROM AUTHOR]

Background: Diffusion-weighted imaging (DWI) has been reported to be a useful technique for diagnosing Creutzfeldt-Jakob disease (CJD). The present study reported DWI results in cases of familial CJD with a V180I mutation (CJD180) in the prion protein gene as well as neurological findings. Methods: A retrospective analysis of 3 patients with V180I was performed. Cerebrospinal fluid (CSF) analysis, brain MRI, single-photon emission computed tomography (SPECT), and magnetic resonance spectroscopy (MRS) were included. CSF was analyzed for biochemical markers, and each patient underwent brain MRI, SPECT, and MRS analysis. A brain biopsy from the frontal cortex, which corresponded to the area of increased DWI signals, was utilized for neuropathological analysis. Results: CSF analysis results revealed elevated total tau protein and the absence of 14-3-3 protein, as well as decreased concentrations of neuron-specific enolase, S100 protein, and prostaglandin E2. All patients presented with unique MRI features. Brain biopsy showed severe spongiform morphology, but comparatively preserved neurons and mild astrocytic gliosis. Accumulations of PrPSc were not detected using the 3F4 antibody, and microglial activation was subtle. SPECT revealed hypoperfusion throughout both hemispheres. MRS revealed a reduced N-acetyl aspartate/creatine ratio. Conclusion: Results from this study suggested that increased DWI signals could reflect severe spongiform changes in CJD180 patients. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: To develop new shuttle vectors for Deinococcus species, the nucleotide sequence of the small cryptic plasmid pUE30 from Deinococcus radiopugnans ATCC19172 was determined. The 2467-bp plasmid possesses two open reading frames, one encoding 88 amino acid residues (Orf1) and the other encoding 501 amino acid residues (Orf2). The predicted amino acid sequence encoded by Orf1 exhibits similarity to the N-terminal regions of replication proteins encoded by repABC-type plasmids of a-proteobacteria. On the other hand, the predicted amino acid sequence encoded by Orf2 exhibits similarity to replication proteins encoded by plasmids of D. radiodurans SARK and Thermus species. Hybrid plasmids consisting of pUE30 and pKatCAT5, which replicates in E. coli with a chloramphenicol resistance determinant, were shown to autonomously replicate in D. grandis ATCC43672. Deletion analysis revealed that Orf2 was necessary for replication of the plasmids in D. grandis. On the other hand, a DNA fragment encompassing the Orf1-coding region was involved in the instability of the plasmid in D. grandis. An expression plasmid that possesses the D. radiodurans minimal groE promoter was constructed, and a firefly luciferase gene was successfully expressed in D. grandis. The D. grandis host-vector system developed in this study should prove useful in the bioremediation of radioactive waste and for the investigation of DNA repair mechanisms. [Copyright &y& Elsevier]

Deinococcus radiodurans possesses a DNA damage response mechanism that acts via the PprI protein to induce RecA and PprA proteins, both of which are necessary in conferring extreme radioresistance. In an effort to further delineate the nature of the DNA damage response mechanism in D. radiodurans, we set out to identify novel components of the PprI-dependent signal transduction pathway in response to radiation stress. Here we demonstrate the discovery of a novel regulatory protein, PprM (a modulator of the PprI-dependent DNA damage response), which is a homolog of cold shock protein (Csp). Disruption of the pprM gene rendered D. radiodurans significantly sensitive to γ-rays. PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from D. geothermalis and Thermus thermophilus. Purified PprM is present as a homodimer under physiological conditions, as the case with Escherichia coli CspD. The pprA pprM double-disruptant strain exhibited higher sensitivity than the pprA or pprM single disruptant strains, suggesting that PprM regulates other hitherto unknown protein(s) important for radioresistance besides PprA. This study strongly suggests that PprM is involved in the radiation response mediated by PprI in D. radiodurans. [ABSTRACT FROM AUTHOR]

To describe the clinical features of Creutzfeldt-Jakob disease with a substitution of arginine for methionine (M232R substitution) at codon 232 (CJD232) of the prion protein gene (PRNP). We evaluated the clinical and laboratory features of 20 CJD232 patients: age of onset, initial symptoms, duration until becoming akinetic and mute, duration until occurrence of periodic sharp and wave complexes on EEG (PSWC), MRI findings, and the presence of CSF 14-3-3 protein. Immunohistochemically, prion protein (PrP) deposition was studied. None of the patients had a family history of CJD. We recognized two clinical phenotypes: a rapidly progressive type (rapidtype) and a slowly progressive type (slow-type). Out of 20 patients, 15 became akinetic and mute, demonstrated myoclonus, and showed PSWC within a mean duration of 3.1, 2.4, and 2.8 months, respectively (rapid-type). Five showed slowly progressive clinical courses (slow-type). Five became akinetic and mute and four demonstrated myoclonus within a mean duration of 20.6 and 15.3 months, respectively, which were significantly longer than those in the rapid-type. Only one demonstrated PSWC 13 months after the onset. Diffuse synaptic-type deposition was demonstrated in four rapidtype patients, and perivacuolar and diffuse synaptic-type deposition in two, and diffuse synaptic-type deposition in one slow-type patient. Three of 50 suspected but non-CJD patients had the M232R substitution. Patients with CJD232 had no family history like patients with sCJD, and showed two different clinical phenotypes in spite of having the same PRNP genotype. More studies are needed to determine whether M232R substitution causes the disease and influences the disease progression. [ABSTRACT FROM AUTHOR]

Background: We have recently begun to doubt the effectiveness of periodic sharp wave complexes observed on electroencephalographs and the detection of 14-3-3 protein in cerebrospinal fluid (CSF) as diagnostic criteria for Creutzfeldt-Jakob disease (CJD). Diffusion-weighted magnetic resonance imaging (DWI) and the detection of total tau (t-tau) protein in CSF may be more sensitive diagnostic criteria. Methods: Among 44 CJD patients, we selected 21 subjects that suffered from early-stage CJD, which was defined as cases in the 6 weeks following the onset of the disease. The sensitivities of DWI and electroencephalographs, as well as those of t-tau protein, 14-3-3 protein, neuron-specific enolase (NSE), and S-100b protein in CSF were compared as diagnostic markers for early-stage CJD. Results: NSE, S-100b protein, t-tau protein, and 14-3-3 protein were detected in the samples from 57.1, 4.8, 95.2, and 76.2% of the 21 early-stage CJD patients, respectively. Additionally, DWI was used to positively identify 90.5% of these cases. Conclusion: We concluded that t-tau protein was the most sensitive of the diagnostic markers for CJD. Moreover, the data in this study showed that detection of t-tau protein combined with DWI identified 98% of the early-stage cases, and these tests should be included as diagnostic criteria for CJD. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: There are currently no markers for evaluating chronological changes in Creutzfeldt-Jakob disease (CJD). We examined if chronological changes in biochemical markers in cerebrospinal fluid (CSF) and diffusion-weighted magnetic resonance imaging (DWI) were utilizable for this purpose. Methods: Ten independent patients were divided into two groups of 5 patients each. We analyzed CSF biochemical markers, DWI and the clinical course in one group. In the remaining group, only the CSF biochemical markers were analyzed before and after the onset of akinetic mutism. Results: The level of total tau (t-tau) protein in CSF in the early phase after disease onset was 2,655 ± 423.9 pg/ml, reaching a mean peak of 14,675 ± 1,240 pg/ml in the middle phase and gradually declining after that. Just before patients deteriorated into akinetic mutism, t-tau protein titers reached a maximum (8,786 ± 2,975 pg/ml). There were dramatic changes in t-tau protein levels throughout the clinical course, unlike the other markers. DWI was not always utilizable, because of discordance with clinical symptoms seen in this study. Four cases exhibited peaks in t-tau protein levels while the patients fell into akinetic mutism except 1 case. Conclusion: Our results suggest that t-tau protein is the most sensitive marker of disease progression in CJD patients. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: In a previous study, we identified the novel protein PprA that plays a critical role in the radiation resistance of Deinococcus radiodurans. In this study, we focussed on the ability of PprA protein to recognize and bind to double-stranded DNA carrying strand breaks, and attempted to visualize radiation-induced DNA strand breaks in mammalian cultured cells by employing PprA protein using an immunofluorescence technique. Increased PprA protein binding to CHO-K1 nuclei immediately following irradiation suggests the protein is binding to DNA strand breaks. By altering the cell permeabilization conditions, PprA protein binding to CHO-K1 mitochondria, which is probably resulted from DNA strand break immediately following irradiation, was also detected. The method developed and detailed in this study will be useful in evaluating DNA damage responses in cultured cells, and could also be applicable to genotoxic tests in the environmental and pharmaceutical fields. [Copyright &y& Elsevier]

Abstract: In a previous study, we identified a novel radiation-inducible protein PprA that plays a critical role in the radiation resistance of Deinococcus radiodurans [Narumi, I., Satoh, K., Cui, S., Funayama, T., Kitayama, S., Watanabe, H., 2004. PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation. Mol. Microbiol. 54, 278–285.]. Despite the interest in mechanisms underlying radiation responses in D. radiodurans, little is known about the radiation responsive promoter for radiation-inducible proteins. In this study, three transcriptional start points for pprA mRNA were identified by primer extension analysis, located at positions −156, −154 and −22 upstream from the pprA translation initiation site. The amount of the three extended products increased in cells exposed to 2 kGy followed by a 0.5-h post-incubation. This suggested the existence of at least two radiation responsive promoters for pprA expression. Functional characterization of the upstream region of the pprA gene using a luciferase reporter assay revealed that the distal promoter is located between positions −208 and −156 from the translation initiation site, while the proximal promoter is located between positions −57 and −22. The region located between positions −57 and −38 was indispensable for proximal promoter activity. Site-directed mutagenesis of a thymine positioned at −33 resulted in severe impairment of promoter activity, and suggested that the thymine functions as a master base for the proximal radiation responsive promoter. The product of the D. radiodurans pprI gene is thought to be a general switch in the radiation response [Hua, Y., Narumi, I., Gao, G., Tian, B., Satoh, K., Kitayama, S., Shen, B., 2003. PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans. Biochem. Biophys. Res. Commun. 306, 354–360.]. We examined the effect of pprI disruption on pprA promoter activity. The results suggested that up-regulation of pprA expression by the pprI gene product is triggered at the promoter level. [Copyright &y& Elsevier]

The extraordinary radiation resistance ofDeinococcus radioduransresults from the efficient capacity of the bacterium to repair DNA double-strand breaks. By analysing the DNA damage repair-deficient mutant, KH311, a unique radiation-inducible gene (designatedpprA) responsible for loss of radiation resistance was identified. Investigationsin vitroshowed that the gene product ofpprA(PprA) preferentially bound to double-stranded DNA carrying strand breaks, inhibitedEscherichia coliexonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATP-dependent and NAD-dependent DNA ligases. These results suggest thatD. radioduranshas a radiation-induced non-homologous end-joining repair mechanism in which PprA plays a critical role. [ABSTRACT FROM AUTHOR]

Investigates the involvement of LexA protein in RecA induction of Deinococcus radiodurans. Factors involved in SOS regulon depression; Impact of RecA-mediated cleavage in deinococcal RecA regulation; Changes in intracellular protein levels after gamma irradiation.

Ionizing radiation induces genetic variations in plants, which makes it useful for plant breeding. A theory that the induced mutations occur randomly in the genome has long been accepted, but is now controversial. Nevertheless, a comparative analysis of the mutations at multiple loci has not been conducted using irradiated M1 genomes that contain all types of mutations. In this study, we identified Arabidopsis mutants (pab2 and pab3) in a mutagenized population of an anthocyanin-positive seed mutant (ban). Both pab2 and pab3 were revealed to be double mutants (tt4 ban and tt8 ban, respectively) that produced similar anthocyanin-less immature seeds, but differentially colored mature seeds. These features enabled the seed color-based detection of de novo M1 mutations in TT4 or TT8 following the irradiation of double heterozygous plants (TT4/tt4 TT8/tt8 ban/ban). Most of the irradiated double heterozygous plants produced anthocyanin-positive immature seeds, but 19 plants produced anthocyanin-less immature seeds. Of these 19 mutants, 2 and 17 exhibited tt4- and tt8-type mature seed coloration, respectively. The molecular analysis of the seed coat DNA from randomly selected anthocyanin-less seeds detected mutations at the locus predicted on the basis of the phenotype. Thus, the simple system developed in this study can reliably detect radiation-induced mutations at multiple loci in irradiated Arabidopsis M1 plants. [ABSTRACT FROM AUTHOR]

Streptomyces lividans TK23 interacts with mycolic acid-containing bacteria (MACB), such as Tsukamurella pulmonis TP-B0596, and this direct cell contact activates its secondary metabolism (e.g., the production of undecylprodigiosin: RED). Here, we employed carbon (12C5+) ion beam-induced mutagenesis to investigate the signature of induced point mutations and further identify the gene(s) responsible for the production of secondary metabolites induced by T. pulmonis. We irradiated spores of the Streptomyces coelicolor strain JCM4020 with carbon ions to generate a mutant library. We screened the RED production-deficient mutants of S. coelicolor by mixing them with T. pulmonis TP-B0596 on agar plates, identifying the red/white phenotype of the growing colonies. Through this process, we selected 59 RED-deficient mutants from around 152,000 tested spores. We resequenced the genomes of 16 mutants and identified 44 point mutations, which revealed the signatures induced by 12C5+-irradiation. Via gene complementation experiments, we also revealed that two genes—glutamate synthase (gltB) and elongation factor G (fusA)—are responsible for the reduced production of RED. [ABSTRACT FROM AUTHOR]

DNA damage response A protein (DdrA) from Deinococcus radiodurans has been suggested to be involved in DNA-repair processes through binding to 3′-ends of single-stranded DNA, thereby protecting the ends from nuclease digestion. In this study, a recombinant C-terminally truncated form of D. radiodurans DdrA (DdrA157) comprising the first 157 residues of DdrA was expressed in Escherichia coli, purified and crystallized. Single crystals of DdrA157 were obtained by the hanging-drop method at 293 K. The crystal belonged to the monoclinic space group P21, with unit-cell parameters a = 46.31, b = 180.26, c = 114.17 Å, β = 90.02°. The crystal was expected to contain 14 molecules in the asymmetric unit. Diffraction data were collected to 2.35 Å resolution on beamline BL-5 at Photon Factory and initial phase determinations were attempted by the molecular-replacement method using the human Rad52 structure. [ABSTRACT FROM AUTHOR]

Human prion diseases are neurodegenerative disorders caused by prion protein. Although infectivity was historically detected only in the central nervous system and lymphoreticular tissues of patients with sporadic Creutzfeldt-Jakob disease, recent reports suggest that the seeding activity of Creutzfeldt-Jakob disease prions accumulates in various non-neuronal organs including the liver, kidney, and skin. Therefore, we reanalyzed autopsy samples collected from patients with sporadic and genetic human prion diseases and found that seeding activity exists in almost all digestive organs. Unexpectedly, activity in the esophagus reached a level of prion seeding activity close to that in the central nervous system in some CJD patients, indicating that the safety of endoscopic examinations should be reconsidered. [ABSTRACT FROM AUTHOR]

Background: Human prion diseases (HPDs) are fatal neurodegenerative disorders characterized by abnormal prion proteins (PrPSc). However, the detection of prion seeding activity in patients with high sensitivity remains challenging. Even though real-time quaking-induced conversion (RT-QuIC) assay is suitable for detecting prion seeding activity in a variety of specimens, it shows lower accuracy when whole blood, blood plasma, and blood-contaminated tissue samples are used. In this study, we developed a novel technology for the in vitro amplification of abnormal prion proteins in HPD to the end of enabling their detection with high sensitivity known as the enhanced quaking-induced conversion (eQuIC) assay. Methods: Three antibodies were used to develop the novel eQUIC method. Thereafter, SD50 seed activity was analyzed using brain tissue samples from patients with prion disease using the conventional RT-QUIC assay and the novel eQUIC assay. In addition, blood samples from six patients with solitary prion disease were analyzed using the novel eQuIC assay. Results: The eQuIC assay, involving the use of three types of human monoclonal antibodies, showed approximately 1000-fold higher sensitivity than the original RT-QuIC assay. However, when this assay was used to analyze blood samples from six patients with sporadic human prion disease, no prion activity was detected. Conclusion: The detection of prion seeding activity in blood samples from patients with sporadic prion disease remains challenging. Thus, the development of alternative methods other than RT-QuIC and eQuIC will be necessary for future research. [ABSTRACT FROM AUTHOR]

The article presents a case study of a 68-year-old male who requested neurological and magnetic resonance imaging (MRI) examinations following the death of his sister due to subarachnoid haemorrhage. The patient was having a difficulty using his right hand, the first symptom of Creutzfeld Jakob disease (CJD), until his condition progressed over time. The patient has undergone a diffusion-weighed MR imaging (DWI), which proved he has CJD condition.

The ubiquitin‒proteasome system (UPS) and autophagy are the two primary cellular pathways of misfolded or damaged protein degradation that maintain cellular proteostasis. When the proteasome is dysfunctional, cells compensate for impaired protein clearance by activating aggrephagy, a type of selective autophagy, to eliminate ubiquitinated protein aggregates; however, the molecular mechanisms by which impaired proteasome function activates aggrephagy remain poorly understood. Here, we demonstrate that activation of aggrephagy is transcriptionally induced by the transcription factor NRF1 (NFE2L1) in response to proteasome dysfunction. Although NRF1 has been previously shown to induce the expression of proteasome genes after proteasome inhibition (i.e., the proteasome bounce-back response), our genome-wide transcriptome analyses identified autophagy-related p62/SQSTM1 and GABARAPL1 as genes directly targeted by NRF1. Intriguingly, NRF1 was also found to be indispensable for the formation of p62-positive puncta and their colocalization with ULK1 and TBK1, which play roles in p62 activation via phosphorylation. Consistently, NRF1 knockdown substantially reduced the phosphorylation rate of Ser403 in p62. Finally, NRF1 selectively upregulated the expression of GABARAPL1, an ATG8 family gene, to induce the clearance of ubiquitinated proteins. Our findings highlight the discovery of an activation mechanism underlying NRF1-mediated aggrephagy through gene regulation when proteasome activity is impaired. [ABSTRACT FROM AUTHOR]

The ubiquitin‒proteasome system (UPS) and autophagy are the two primary cellular pathways of misfolded or damaged protein degradation that maintain cellular proteostasis. When the proteasome is dysfunctional, cells compensate for impaired protein clearance by activating aggrephagy, a type of selective autophagy, to eliminate ubiquitinated protein aggregates; however, the molecular mechanisms by which impaired proteasome function activates aggrephagy remain poorly understood. Here, we demonstrate that activation of aggrephagy is transcriptionally induced by the transcription factor NRF1 (NFE2L1) in response to proteasome dysfunction. Although NRF1 has been previously shown to induce the expression of proteasome genes after proteasome inhibition (i.e., the proteasome bounce-back response), our genome-wide transcriptome analyses identified autophagy-related p62/SQSTM1 and GABARAPL1 as genes directly targeted by NRF1. Intriguingly, NRF1 was also found to be indispensable for the formation of p62-positive puncta and their colocalization with ULK1 and TBK1, which play roles in p62 activation via phosphorylation. Consistently, NRF1 knockdown substantially reduced the phosphorylation rate of Ser403 in p62. Finally, NRF1 selectively upregulated the expression of GABARAPL1, an ATG8 family gene, to induce the clearance of ubiquitinated proteins. Our findings highlight the discovery of an activation mechanism underlying NRF1-mediated aggrephagy through gene regulation when proteasome activity is impaired. [ABSTRACT FROM AUTHOR]

Abstract Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common form of human prion disease; it is characterized as a transmissible spongiform encephalopathy. Previously, only two case reports have discussed crossed cerebellar diaschisis (CCD) and sCJD. Herein, we detected CCD in five patients with sCJD and four patients with genetic CJD through SPECT. The combined data of the nine patients further supported CCD when analyzed by 3D-SSP. CCD can occur in several disorders, including, stroke, epilepsy, and encephalitis. Hence, CCD possibly could help in making earlier CJD diagnoses. Further studies with more patients are necessary to confirm the strong relationship here suggested between CJD and CCD. [ABSTRACT FROM AUTHOR]

Background: The gamma-isoform of the 14-3-3 protein (14-3-3 gamma) is expressed in neurons, and could be a specific marker for neuronal damage. This protein has been reported as a detectable biomarker, especially in the cerebrospinal fluid (CSF) of Creutzfeldt-Jakob disease (CJD) patients by Western blotting (WB) or enzyme-linked immunosorbent assays (ELISAs). Western blotting for 14-3-3 gamma is not sensitive, and the reported data are conflicting among publications. An ELISA specific for 14-3-3 gamma is not available.Methods: CJD patients (n=114 sporadic CJD patients, 7 genetic CJD, and 3 iatrogenic CJD) and 99 patients with other neurodegenerative diseases were examined in this study. The CSF samples obtained were analyzed by Western blotting for 14-3-3 gamma, and by ELISA for total tau protein. We evaluated the sensitivity and specificity of the newly developed sandwich ELISA for 14-3-3 gamma.Results: The cut-off value of the 14-3-3 gamma ELISA was >1, 683 AU/ml; and sensitivity was 95.2%, with 72.7% specificity. This specificity was the same for the total tau protein ELISA. Seven CJD cases were negative by WB but positive using the 14-3-3 gamma ELISA, indicating that the ELISA is more sensitive. All 21 cases of early stage CJD could be diagnosed using a combination of the 14-3-3γ ELISA and diffusion weighted MR imaging (DWI-MRI).Conclusion: The 14-3-3 gamma ELISA was more sensitive than conventional WB, and was useful for laboratory diagnosis of CJD, similar to the ELISA for the tau protein. Using DWI-MRI and these ELISA tests on CSF, diagnosis of CJD will be possible even at early stages of the disease. [ABSTRACT FROM AUTHOR]

Stem cell therapy for ischemic stroke holds great promise for the treatment of neurological impairment and has moved from the laboratory into early clinical trials. The mechanism of action of stem cell therapy includes the bystander effect and cell replacement. The bystander effect plays an important role in the acute to subacute phase, and cell replacement plays an important role in the subacute to chronic phase. Intraarterial (IA) transplantation is less invasive than intraparenchymal transplantation and can provide more cells in the affected brain region than intravenous transplantation. However, transplanted cell migration was reported to be insufficient, and few transplanted cells were retained in the brain for an extended period. Therefore, the bystander effect was considered the main mechanism of action of IA stem cell transplantation. In most clinical trials, IA transplantation was performed during the acute and subacute phases. Although clinical trials of IA transplantation demonstrated safety, they did not demonstrate satisfactory efficacy in improving patient outcomes. To increase efficacy, increased migration of transplanted cells and production of long surviving and effective stem cells would be crucial. Given the lack of knowledge on this subject, we review and summarize the mechanisms of action of transplanted stem cells and recent advancements in preclinical and clinical studies to provide information and guidance for further advancement of acute/subacute phase IA stem cell transplantation therapy for ischemic stroke. [ABSTRACT FROM AUTHOR]

Cell transplantation therapy for cerebral infarction has emerged as a promising treatment to reduce brain damage and enhance functional recovery. We previously reported that intra-arterial delivery of bone marrow mesenchymal stem cells (MSCs) enables superselective cell administration to the infarct area and results in significant functional recovery after ischemic stroke in a rat model. However, to reduce the risk of embolism caused by the transplanted cells, an optimal cell number should be determined. At 24 h after middle cerebral artery occlusion and reperfusion, we administered human MSCs (low dose: 1 × 10 cells; high dose: 1 × 10 cells) and then assessed functional recovery, inflammatory responses, cell distribution, and mortality. Rats treated with high- or low-dose MSCs showed behavioral recovery. At day 8 post-stroke, microglial activation was suppressed significantly, and interleukin (IL)-1β and IL-12p70 were reduced in both groups. Although high-dose MSCs were more widely distributed in the cortex and striatum of rats, the degree of intravascular cell aggregation and mortality was significantly higher in the high-dose group. In conclusion, selective intra-arterial transplantation of low-dose MSCs has anti-inflammatory effects and reduces the adverse effects of embolic complication, resulting in sufficient functional recovery of the affected brain. [ABSTRACT FROM AUTHOR]

The application of entomopathogenic fungi such as Isaria fumosorosea to combat insect pests on plants is complicated by their sensitivity to commonly used fungicides. In this study, I. fumosorosea mutants with enhanced resistance to the fungicide benomyl were induced by irradiation using either ion beams or gamma rays, or a combination of the two. When grown on agar containing benomyl, mycelial growth was observed for five of the six mutant isolates at benomyl concentrations that were more than 2000-fold those observed for the wild-type isolate ( EC50: > 5000 mg L−1 c.f. EC50: 2.5 mg L−1 for the wild-type isolate). The mutant isolates evaluated also showed enhanced resistance to other fungicides at recommended field application rates. No differences were observed at the β-tubulin locus between the wild-type and the mutant isolates, suggesting that the enhanced benomyl resistance was not attributable to mutations in that gene. Ion beams and gamma rays are thus potentially useful tools for inducing beneficial fungal mutations and thereby improving the potential for application of entomopathogenic fungi as microbial control agents. [ABSTRACT FROM AUTHOR]

A national system for surveillance of prion diseases (PrDs) was established in Japan in April 1999. Here, we analyzed the relationships among prion protein gene (PRNP) mutations and the clinical features, cerebrospinal fluid (CSF) markers, and pathological characteristics of the major genotypes of genetic PrDs (gPrDs). We retrospectively analyzed age at onset and disease duration; the concentrations and incidences of 14-3-3 protein, tau protein, and abnormal prion protein (PrPSc) in the CSF of 309 gPrD patients with P102L, P105L, E200K, V180I, or M232R mutations; and brain pathology in 32 autopsied patients. Three clinical phenotypes were seen: rapidly progressive Creutzfeldt-Jakob disease (CJD), which included 100% of E200K cases, 70% of M232R, and 21% of P102L; slowly progressive CJD, which included 100% of V180I and 30% of M232R; and Gerstmann-Sträussler-Scheinker disease, which included 100% of P105L and 79% of P102L. PrPSc was detected in the CSF of more than 80% of patients with E200K, M232R, or P102L mutations but in only 39% of patients with V180I. V180I was accompanied by weak PrP immunoreactivity in the brain. Patients negative for PrPSc in the CSF were older at disease onset than positive patients. Patients with mutations associated with high 14-3-3 protein levels in the CSF typically had synaptic deposition of PrP in the brain and a rapid course of disease. The presence of small PrP protein fragments in brain homogenates was not correlated with other clinicopathological features. Positivity for PrPSc in the CSF may reflect the pathological process before or at disease onset, or abnormality in the secretion or metabolism of PrPSc. The amount of 14-3-3 protein in the CSF likely indicates the severity of the pathological process and accompanying neuronal damage. These characteristic features of the CSF in cases of gPrD will likely facilitate accurate diagnosis and clinicopathological study of the various disease subtypes. [ABSTRACT FROM AUTHOR]

The objective was to characterize a rapidly progressive subtype of Alzheimer's disease (rpAD). Multicenter (France, Germany, Japan, Spain) retrospective analyses of neuropathologically confirmed rpAD cases initially classified as prion disease due to their clinical phenotype were performed. Genetic properties, cerebrospinal fluid biomarkers, neuropathology, and clinical features were examined. Eighty-nine patients were included (median survival 10 months). APOE and PRNP codon 129 genotype distribution paralleled a healthy control group. APOE ℇ4 homozygosity was absent. Cerebrospinal fluid biomarkers were abnormal, but within a range as expected for classic AD, except for proteins 14-3-3, which were detectable in 42%. Thus, evidence of the existence of rpAD is accumulating. The APOE profile is intriguing, suggesting that this very rapid disease form might represent a distinct subtype of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Abstract: A DNA repair-promoting protein, PprA, was isolated from a radiation resistant bacterium, Deinococcus radiodurans [I. Narumi, K. Sato, S. Cui, T. Funayama, S. Kitayama, H. Watanabe, PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation, Mol. Microbiol. 54 (2004) 278–285]. Despite several studies, however, the function of PprA is not still clear. We used atomic force microscopy (AFM) to elucidate the role of this protein in the DNA repair pathway. In the present study, interaction between the linear DNA and PprA protein was imaged and analyzed by AFM without any fixation or staining. Though both end-bound and internally bound PprA was observed, the affinity of the end-bound protein was greater considering the proportion of features of binding analyzed by AFM. In some conditions, looping forms of the DNA–PprA complex were observed. Gel filtration high performance liquid chromatography (HPLC) was also conducted to estimate the molecular weight of this protein. The result of the HPLC analysis suggested that PprA formed multimers in buffer solution without DNA. [Copyright &y& Elsevier]

Serine 129 (S129) phosphorylation of α-synuclein (αSyn) is a central feature of Lewy body (LB) disease pathology. Although the neighboring tyrosine residues Y125, Y133, and Y136 are also phosphorylation sites, little is known regarding potential roles of phosphorylation cross-talk between these sites and its involvement in the pathogenesis of LB disease. Here, we found that αSyn aggregates are predominantly phosphorylated at Y136 in the Lewy body dementia brain, which is mediated by unexpected kinase activity of Casein kinase 2 (CK2). Aggregate formation with S129 and Y136 phosphorylation of recombinant αSyn (r-αSyn) were induced by CK2 but abolished by replacement of S129 with alanine (S129A) in vitro. Mutation of Y136 to alanine (Y136A) promoted aggregate formation and S129 phosphorylation of r-αSyn by CK2 in vitro. Introduction of Y136A r-αSyn oligomers into cultured cells exhibited increased levels of aggregates with S129 phosphorylation compared to wild-type r-αSyn oligomers. In addition, aggregate formation with S129 phosphorylation induced by introduction of wild-type r-αSyn oligomers was significantly attenuated by CK2 inhibition, which resulted in an unexpected increase in Y136 phosphorylation in cultured cells. Our findings suggest the involvement of CK2-related αSyn Y136 phosphorylation in the pathogenesis of LB disease and its potential as a therapeutic target. [ABSTRACT FROM AUTHOR]

Light/dark cycling is an inherent condition of outdoor microalgae cultivation, but is often unfavorable for lipid accumulation. This study aims to identify promising targets for metabolic engineering of improved lipid accumulation under outdoor conditions. Consequently, the lipid-rich mutant Chlamydomonas sp. KOR1 was developed through light/dark-conditioned screening. During dark periods with depressed CO2 fixation, KOR1 shows rapid carbohydrate degradation together with increased lipid and carotenoid contents. KOR1 was subsequently characterized with extensive mutation of the ISA1 gene encoding a starch debranching enzyme (DBE). Dynamic time-course profiling and metabolomics reveal dramatic changes in KOR1 metabolism throughout light/dark cycles. During light periods, increased flux from CO2 through glycolytic intermediates is directly observed to accompany enhanced formation of small starch-like particles, which are then efficiently repartitioned in the next dark cycle. This study demonstrates that disruption of DBE can improve biofuel production under light/dark conditions, through accelerated carbohydrate repartitioning into lipid and carotenoid. Kato et al. describe the isolation of a mutant Chlamydomonas sp. KOR1 that accumulates lipid even during diurnal cycles and has extensive mutations in the ISA1 gene encoding a starch debranching enzyme (DBE). They further demonstrate that disruption of DBE can improve biofuel production under light/dark conditions, through accelerated carbohydrate repartitioning into lipid and carotenoid. [ABSTRACT FROM AUTHOR]

We report the general autopsy findings of abnormal prion protein (PrP) deposits with their seeding activities, as assessed by the real‐time quaking‐induced conversion (RT‐QuIC) method, in a 72‐year‐old female patient with sporadic Creutzfeldt–Jakob disease (sCJD). At 68 years of age, she presented with gait disturbance and visual disorders. Electroencephalography showed periodic synchronous discharge. Myoclonus was also observed. A genetic test revealed that PRNP codon 129 was methionine/methionine (MM). She died of pneumonia three years and four months after disease onset, and a general autopsy was performed. The brain weighed 650 g and appeared markedly atrophic. Immunohistochemistry for PrP revealed synaptic PrP deposits and coarse PrP deposits in the cerebral cortices, basal ganglia, cerebellum, and brainstem. Western blot analysis identified type 1 proteinase‐K‐resistant PrP in frontal cortex samples. PrP deposits were also observed in systemic organs, including the femoral nerve, psoas major muscle, abdominal skin, adrenal medulla, zona reticularis of the adrenal gland, islet cells of the pancreas, and thyroid gland. The RT‐QuIC method revealed positive seeding activities in all examined organs, including the frontal cortex, femoral nerve, psoas major muscle, scalp, abdominal skin, adrenal gland, pancreas, and thyroid gland. The following 50% seeding dose (SD50) values were 9.5 (frontal cortex); 8 ± 0.53 (femoral nerve); 7 ± 0.53 (psoas major muscle); and 7.88 ± 0.17 (scalp). The SD50 values for the adrenal gland, dermis, pancreas, and thyroid gland were 6.12 ± 0.53, 5.25, 4.75, and 4.5, respectively. PrP deposits in general organs may be associated with long‐term disease duration. This case indicated the necessity for general autopsies in sCJD cases to establish strict infection control procedures for surgical treatment and to examine certain organs. [ABSTRACT FROM AUTHOR]

Sporadic Creutzfeldt-Jakob disease is a fatal neurodegenerative disease caused by misfolded prion proteins (PrPSc). Effective therapeutics are currently not available and accurate diagnosis can be challenging. Clinical diagnostic criteria use a combination of characteristic neuropsychiatric symptoms, CSF proteins 14-3-3, MRI, and EEG. Supportive biomarkers, such as high CSF total tau, could aid the diagnostic process. However, discordant studies have led to controversies about the clinical value of some established surrogate biomarkers. Development and clinical application of disease-specific protein aggregation and amplification assays, such as real-time quaking induced conversion (RT-QuIC), have constituted major breakthroughs for the confident pre-mortem diagnosis of sporadic Creutzfeldt-Jakob disease. Updated criteria for the diagnosis of sporadic Creutzfeldt-Jakob disease, including application of RT-QuIC, should improve early clinical confirmation, surveillance, assessment of PrPSc seeding activity in different tissues, and trial monitoring. Moreover, emerging blood-based, prognostic, and potentially pre-symptomatic biomarker candidates are under investigation. [ABSTRACT FROM AUTHOR]

All theories related to the evolution of Deinococcus radiodurans have a common denominator: the strong positive correlation between ionizing-radiation resistance and desiccation tolerance. Currently, the widespread hypothesis is that D. radiodurans' ionizing-radiation resistance is a consequence of this organism's adaptation to desiccation (desiccation adaptation hypothesis). Here, we draw attention to major discrepancy that has emerged between the ''desiccation adaptation hypothesis'' and recent findings in computational biology, experimental research, and terrestrial subsurface surveys. We explain why the alternative hypothesis, suggesting that D. radiodurans' desiccation tolerance could be a consequence of this organism's adaptation to ionizing radiation (radiation adaptation hypothesis), should be considered on equal basis with the ''desiccation adaptation hypothesis''. [ABSTRACT FROM AUTHOR]

The original version of this article unfortunately contained an error in affiliation of Yuhtaka Fukuda. [ABSTRACT FROM AUTHOR]