Detection and characterization of genome-wide mutations in M1 vegetative cells of gamma-irradiated Arabidopsis.

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.

Detection limitations of prion seeding activities in blood samples from patients with sporadic prion disease.

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.

Formalin RT-QuIC assay detects prion-seeding activity in formalin-fixed brain samples from sporadic Creutzfeldt-Jakob disease patients.

BACKGROUND: The neuropathology of sporadic Creutzfeldt-Jakob disease (sCJD) is usually investigated using formalin-fixed and formic acid-treated brain tissue. However, formalin and formic acid treatment can interfere with immunostaining of abnormal prion protein. Therefore, there is a need for biochemical methods other than immunostaining to investigate abnormal prion protein in postmortem tissue. We developed RT-QuIC to quantitate the seeding activity (SD50) of sCJD brain tissue treated with formalin and formic acid. METHODS: We used endpoint RT-QuIC assays to analyze SD50 in formalin-fixed brain tissue from 19 sCJD patients (14 MM1 cases, 3 MM2-thalamic form [MM2T] cases and 2 MM2-cortical form [MM2C] cases) diagnosed according to Parchi's classification. We assessed SD50 in brains after incubation in formalin solution for over 1 month, and after treating formalin-fixed brain tissue with formic acid. We also examined how the SD50 values from formalin-fixed brain samples compared with neuropathological and immunohistochemical findings. RESULTS: The SD50 values of formalin-fixed brain samples from 14 MM1 cases, 2 MM2C cases, and 2 MM2T cases were 107.77±0.57/g tissue, 107.44±0.24/g tissue and 106.00±0.77/g tissue, respectively. The average SD50 value in MM1 unfixed brains decreased by 102.04 after formalin fixation for 1 month. In MM1 cases, after combined formalin and formic acid treatment, the SD50 value was reduced by approximately 105.16 compared with that of unfixed tissue. The SD50 values of formalin-fixed tissue showed a consistent pattern with the neuropathological findings in most brain regions examined. CONCLUSION: RT-QuIC enables the study of formalin-fixed brain tissue from sCJD patients that has not previously been amenable to analysis.

Abnormal prion protein deposits with high seeding activities in the skeletal muscle, femoral nerve, and scalp of an autopsied case of sporadic Creutzfeldt-Jakob disease.

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.

Prion protein interacts with the metabotropic glutamate receptor 1 and regulates the organization of Ca2+ signaling.

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.

MM2-type sporadic Creutzfeldt-Jakob disease: new diagnostic criteria for MM2-cortical type.

OBJECTIVE: To clinically diagnose MM2-cortical (MM2C) and MM2-thalamic (MM2T)-type sporadic Creutzfeldt-Jakob disease (sCJD) at early stage with high sensitivity and specificity. METHODS: We reviewed the results of Creutzfeldt-Jakob disease Surveillance Study in Japan between April 1999 and September 2019, which included 254 patients with pathologically confirmed prion diseases, including 9 with MM2C-type sCJD (MM2C-sCJD) and 10 with MM2T-type sCJD (MM2T-sCJD), and 607 with non-prion diseases. RESULTS: According to the conventional criteria of sCJD, 4 of 9 patients with MM2C- and 7 of 10 patients with MM2T-sCJD could not be diagnosed with probable sCJD until their death. Compared with other types of sCJD, patients with MM2C-sCJD showed slower progression of the disease and cortical distribution of hyperintensity lesions on diffusion-weighted images of brain MRI. Patients with MM2T-sCJD also showed relatively slow progression and negative results for most of currently established investigations for diagnosis of sCJD. To clinically diagnose MM2C-sCJD, we propose the new criteria; diagnostic sensitivity and specificity to distinguish 'probable' MM2C-sCJD from other subtypes of sCJD, genetic or acquired prion diseases and non-prion disease controls were 77.8% and 98.5%, respectively. As for MM2T-sCJD, clinical and laboratory features are not characterised enough to develop its diagnostic criteria. CONCLUSIONS: MM2C-sCJD can be diagnosed at earlier stage using the new criteria with high sensitivity and specificity, although it is still difficult to diagnose MM2T-sCJD clinically.

Correction to: Intra-Arterial Transplantation of Low-Dose Stem Cells Provides Functional Recovery Without Adverse Effects After Stroke.

The original version of this article unfortunately contained an error in affiliation of Yuhtaka Fukuda.

Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Pleiotropic protein promoting DNA repair A (PprA) is a key protein facilitating the extreme radiation resistance of Deinococcus radiodurans. PprA is a unique protein to the genus Deinococcus and exists as an oligomer ranging from a tetramer to an ∼100-mer depending on protein concentrations. Here, the X-ray crystal structure of PprA was determined to clarify how PprA confers radiation resistance. The tertiary structure of dimeric PprA was elucidated by using mutants obtained with random and site-directed mutagenesis methods (W183R and A139R); these mutants have disabled DNA binding and polymerization functions. Because the mutant A139R and W183R proteins have dimeric assemblies with 2 different interfaces (Interfaces 1 and 2), the linear and oligomerized PprA model was constructed as a left-handed face-to-face periodic screw structure. In addition, the linear structure in solution was confirmed by small-angle scattering experiments. The site-directed mutational analysis identified essential basic amino acids for DNA binding. These analytical data support the hypothesis that a complex assembly of PprA molecules, which are extended and have a screw structure, surrounds and stretches the DNA strand, acting as a novel guide to colocalize the DNA strands for efficient DNA repairs.-Adachi, M., Shimizu, R., Shibazaki, C., Satoh, K., Fujiwara, S., Arai, S., Narumi, I., Kuroki, R. Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Concurrent Validity of the ABC Dementia Scale with Other Standard Scales: A New Comprehensive Instrument for Assessing Dementia in Japan.

BACKGROUND/AIMS: The ABC Dementia Scale (ABC-DS), a new tool for evaluating dementia, was developed in Japan. The ABC-DS is a comprehensive instrument that can simultaneously evaluate activities of daily living (ADLs), behavioral and psychological symptoms of dementia (BPSD), and cognitive function. The ABC-DS can be administered easily and quickly and can clarify the severity of dementia and its changes over time. While the ABC-DS has been reported to be useful in Alzheimer disease (AD)-type dementia, it has not yet been studied in other types of dementia. The purpose of this study was to reevaluate the standard validity of ABC-DS separately for various dementia types and severities. METHODS: We evaluated the ABC-DS in outpatients at 1 hospital in Nagasaki Prefecture and patients who use the facility. Domain A, corresponding to ADLs, correlated with Disability Assessment for Dementia (DAD); domain B, corresponding to BPSD, correlated with the Neuropsychiatric Inventory (NPI); domain C, corresponding to cognitive functions, correlated with Mini-Mental State Examination (MMSE); and the total score of the ABC-DS correlated with the Clinical Dementia Rating (CDR). RESULTS: 102 patients, comprising 38 males and 64 females with an average age of 80.7 ± 8.6 years, were enrolled. AD-type dementia was present in 38 cases, vascular dementia (VaD) in 23, mixed dementia in 23, dementia with Lewy bodies in 6, argyrophilic grain dementia in 9, and mild cognitive impairment in 3. A strong correlation was found between ABC-DS domain scores and their respective reference neuropsychological instruments (domain A and the DAD, domain B and the NPI, domain C and the MMSE, and total score and CDR). The correlation of each ABC-DS domain score with the corresponding standard scale depended on the type and severity of dementia, and we observed moderate or high correlations in AD and VaD patients with moderate and severe dementia. DISCUSSION: Although the ABC-DS targets AD, it can be used in VaD based on the results of this study. In other types of dementia, the results differed depending on the domain; in some conditions, the ABC-DS may not show sufficient concurrent validity with other standard scales. Also, the ABC-DS is more beneficial for moderate-to-severe dementia, as reported in previous studies. It is highly useful in clinical practice in Japan since there more than half of all patients have moderate-to-severe dementia.

Type I interferon protects neurons from prions in in vivo models.

Infectious prions comprising abnormal prion protein, which is produced by structural conversion of normal prion protein, are responsible for transmissible spongiform encephalopathies including Creutzfeldt-Jakob disease in humans. Prions are infectious agents that do not possess a genome and the pathogenic protein was not thought to evoke any immune response. Although we previously reported that interferon regulatory factor 3 (IRF3) was likely to be involved in the pathogenesis of prion diseases, suggesting the protective role of host innate immune responses mediated by IRF3 signalling, this remained to be clarified. Here, we investigated the reciprocal interactions of type I interferon evoked by IRF3 activation and prion infection and found that infecting prions cause the suppression of endogenous interferon expression. Conversely, treatment with recombinant interferons in an ex vivo model was able to inhibit prion infection. In addition, cells and mice deficient in type I interferon receptor (subunit interferon alpha/beta receptor 1), exhibited higher susceptibility to 22L-prion infection. Moreover, in in vivo and ex vivo prion-infected models, treatment with RO8191, a selective type I interferon receptor agonist, inhibited prion invasion and prolonged the survival period of infected mice. Taken together, these data indicated that the interferon signalling interferes with prion propagation and some interferon-stimulated genes might play protective roles in the brain. These findings may allow for the development of new strategies to combat fatal diseases.

Postmortem Quantitative Analysis of Prion Seeding Activity in the Digestive System.

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.

Novel anti-suprabasin antibodies may contribute to the pathogenesis of neuropsychiatric systemic lupus erythematosus.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is often difficult to diagnose and distinguish from other diseases, because no NPSLE-specific antibodies have been identified. We developed a novel proteomic strategy for identifying and profiling antigens in immune complexes in the cerebrospinal fluid (CSF), and applied this strategy to 26 NPSLE patients. As controls, we also included 25 SLE patients without neuropsychiatric manifestations (SLE), 15 with relapsing remitting multiple sclerosis (MS) and 10 with normal pressure hydrocephalus (NPH). We identified immune complexes of suprabasin (SBSN) in the CSF of the NPSLE group. The titer of anti-SBSN antibodies was significantly higher in the CSF of the NPSLE group compared to those of the SLE, MS and NPH groups. Microarray data showed that the senescence and autophagy pathways were significantly changed in astrocytes exposed to anti-SBSN antibodies. Our findings indicate that SBSN could be a novel autoantibody for the evaluation of suspected NPSLE.

Biochemical features of genetic Creutzfeldt-Jakob disease with valine-to-isoleucine substitution at codon 180 on the prion protein gene.

Valine-to-isoleucine substitution at codon 180 of the prion protein gene is only observed in patients with Creutzfeldt-Jakob disease and accounts for approximately half of all cases of genetic prion disease in Japan. In the present study, we investigated the biochemical characteristics of valine-to-isoleucine substitution at codon 180 in the prion protein gene, using samples obtained from the autopsied brains of seven patients with genetic Creutzfeldt-Jakob disease exhibiting this mutation (diagnoses confirmed via neuropathological examination). Among these patients, we observed an absence of diglycosylated and monoglycosylated forms of PrPres at codon 181. Our findings further indicated that the abnormal prion proteins were composed of at least three components, although smaller carboxyl-terminal fragments were predominant. Western blot analyses revealed large amounts of PrPres in the cerebral neocortices, where neuropathological examination revealed marked spongiosis. Relatively smaller amounts of PrPres were detected in the hippocampus, where milder spongiosis was observed, than in the cerebral neocortex. These findings indicate that abnormal prion proteins in the neocortex are associated with severe toxicity, resulting in severe spongiosis. Our findings further indicate that the valine-to-isoleucine substitution is not a polymorphism, but rather an authentic pathogenic mutation associated with specific biochemical characteristics that differ from those observed in sporadic Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease with Alzheimer pathology, presenting with status epilepticus following repeated partial seizures: a case report and literature review.

BACKGROUND: Creutzfeldt-Jakob disease (CJD) is a fatal neurodegenerative disease. Common first symptoms are dementia, cerebellar ataxia, visual disturbance, and psychiatric symptoms. Seizure as the first symptom of CJD is a very rare finding. CASE PRESENTATION: We experienced an elderly woman who presented initially with status epilepticus following repeated partial seizures in the course of Alzheimer disease (AD) dementia. Anti-convulsive therapy had no effect. Autopsy revealed definite CJD with AD pathology. COCLUSIONS: This is the first reported CJD case presenting with status epilepticus in the course of AD dementia.

Prosultiamine for treatment of lower urinary tract dysfunction accompanied by human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis.

OBJECTIVES: To evaluate oral prosultiamine treatment in patients with overactive bladder accompanied by human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis. METHODS: This was a prospective, single-center, open-label study. Patients received oral prosultiamine (300 mg) once daily in the morning, and the overactive bladder symptom score and urine levels of overactive bladder-related biomarkers (nerve growth factor/creatinine and adenosine triphosphate/creatinine) 12 weeks after the initial administration were compared with the baseline values. In addition, the urodynamic parameters, including involuntary detrusor contraction and detrusor sphincter dyssynergia, were evaluated before and after treatment. RESULTS: A total of 16 patients were recruited for this clinical study. In the overactive bladder symptom score, night-time frequency, urgency and the total score improved after oral prosultiamine treatment (P = 0.028, 0.001 and 0.004, respectively). Both urinary nerve growth factor/creatinine and adenosine triphosphate/creatinine levels decreased significantly after the treatment (P = 0.004 and 0.017, respectively). Urodynamic studies showed that the maximum cystometric capacity increased significantly after the treatment. However, the symptoms disappeared because of the treatment in six of 10 patients with involuntary detrusor contraction (60%) and three of seven patients with detrusor sphincter dyssynergia (42.9%). There were no serious adverse events. CONCLUSIONS: The changes in urodynamic parameters and urine levels of overactive bladder-related markers suggest that oral prosultiamine is a safe and effective treatment for overactive bladder with human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis.

Cerebrospinal fluid real-time quaking-induced conversion is a robust and reliable test for sporadic creutzfeldt-jakob disease: An international study.

Real-time quaking-induced conversion (RT-QuIC) has been proposed as a sensitive diagnostic test for sporadic Creutzfeldt-Jakob disease; however, before this assay can be introduced into clinical practice, its reliability and reproducibility need to be demonstrated. Two international ring trials were undertaken in which a set of 25 cerebrospinal fluid samples were analyzed by a total of 11 different centers using a range of recombinant prion protein substrates and instrumentation. The results show almost complete concordance between the centers and demonstrate that RT-QuIC is a suitably reliable and robust technique for clinical practice. Ann Neurol 2016;80:160-165.

DNA Gyrase of Deinococcus radiodurans is characterized as Type II bacterial topoisomerase and its activity is differentially regulated by PprA in vitro.

The multipartite genome of Deinococcus radiodurans forms toroidal structure. It encodes topoisomerase IB and both the subunits of DNA gyrase (DrGyr) while lacks other bacterial topoisomerases. Recently, PprA a pleiotropic protein involved in radiation resistance in D. radiodurans has been suggested for having roles in cell division and genome maintenance. In vivo interaction of PprA with topoisomerases has also been shown. DrGyr constituted from recombinant gyrase A and gyrase B subunits showed decatenation, relaxation and supercoiling activities. Wild type PprA stimulated DNA relaxation activity while inhibited supercoiling activity of DrGyr. Lysine133 to glutamic acid (K133E) and tryptophane183 to arginine (W183R) replacements resulted loss of DNA binding activity in PprA and that showed very little effect on DrGyr activities in vitro. Interestingly, wild type PprA and its K133E derivative continued interacting with GyrA in vivo while W183R, which formed relatively short oligomers did not interact with GyrA. The size of nucleoid in PprA mutant (1.9564 ± 0.324 µm) was significantly bigger than the wild type (1.6437 ± 0.345 µm). Thus, we showed that DrGyr confers all three activities of bacterial type IIA family DNA topoisomerases, which are differentially regulated by PprA, highlighting the significant role of PprA in DrGyr activity regulation and genome maintenance in D. radiodurans.

Conformational properties of prion strains can be transmitted to recombinant prion protein fibrils in real-time quaking-induced conversion.

The phenomenon of prion strains with distinct biological characteristics has been hypothesized to be involved in the structural diversity of abnormal prion protein (PrP(Sc)). However, the molecular basis of the transmission of strain properties remains poorly understood. Real-time quaking-induced conversion (RT-QUIC) is a cell-free system that uses Escherichia coli-derived recombinant PrP (rPrP) for the sensitive detection of PrP(Sc). To investigate whether the properties of various prion strains can be transmitted to amyloid fibrils consisting of rPrP (rPrP fibrils) using RT-QUIC, we examined the secondary structure, conformational stability, and infectivity of rPrP fibrils seeded with PrP(Sc) derived from either the Chandler or the 22L strain. In the first round of the reaction, there were differences in the secondary structures, especially in bands attributed to ß-sheets, as determined by infrared spectroscopy, and conformational stability between Chandler-seeded (1st-rPrP-fib(Ch)) and 22L-seeded (1st-rPrP-fib(22L)) rPrP fibrils. Of note, specific identifying characteristics of the two rPrP fibril types seen in the ß-sheets resembled those of the original PrP(Sc). Furthermore, the conformational stability of 1st-rPrP-fib(Ch) was significantly higher than that of 1st-rPrP-fib(22L), as with Chandler and 22L PrP(Sc). The survival periods of mice inoculated with 1st-rPrP-fib(Ch) or 1st-rPrP-fib(22L) were significantly shorter than those of mice inoculated with mixtures from the mock 1st-round RT-QUIC procedure. In contrast, these biochemical characteristics were no longer evident in subsequent rounds, suggesting that nonspecific uninfected rPrP fibrils became predominant probably because of their high growth rate. Together, these findings show that at least some strain-specific conformational properties can be transmitted to rPrP fibrils and unknown cofactors or environmental conditions may be required for further conservation. Importance: The phenomenon of prion strains with distinct biological characteristics is assumed to result from the conformational variations in the abnormal prion protein (PrP(Sc)). However, important questions remain about the mechanistic relationship between the conformational differences and the strain diversity, including how strain-specific conformations are transmitted. In this study, we investigated whether the properties of diverse prion strains can be transmitted to amyloid fibrils consisting of E. coli-derived recombinant PrP (rPrP) generated by real-time quaking-induced conversion (RT-QUIC), a recently developed in vitro PrP(Sc) formation method. We demonstrate that at least some of the strain-specific conformational properties can be transmitted to rPrP fibrils in the first round of RT-QUIC by examining the secondary structure, conformational stability, and infectivity of rPrP fibrils seeded with PrP(Sc) derived from either the Chandler or the 22L prion strain. We believe that these findings will advance our understanding of the conformational basis underlying prion strain diversity.

Intra-arterial transplantation of low-dose stem cells provides functional recovery without adverse effects after stroke.

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(4) cells; high dose: 1 × 10(6) 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)-1ß 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.