Double-multilayer monochromators for high-energy and large-field X-ray imaging applications with intense pink beams at SPring-8 BL20B2.

In this study, double-multilayer monochromators that generate intense, high-energy, pink X-ray beams are designed, installed and evaluated at the SPring-8 medium-length (215 m) bending-magnet beamline BL20B2 for imaging applications. Two pairs of W/B4C multilayer mirrors are designed to utilize photon energies of 110 keV and 40 keV with bandwidths of 0.8% and 4.8%, respectively, which are more than 100 times larger when compared with the Si double-crystal monochromator (DCM) with a bandwidth of less than 0.01%. At an experimental hutch located 210 m away from the source, a large and uniform beam of size 14 mm (V) × 300 mm (H) [21 mm (V) × 300 mm (H)] was generated with a high flux density of 1.6 × 109 photons s-1 mm-2 (6.9 × 1010 photons s-1 mm-2) at 110 keV (40 keV), which marked a 300 (190) times increase in the photon flux when compared with a DCM with Si 511 (111) diffraction. The intense pink beams facilitate advanced X-ray imaging for large-sized objects such as fossils, rocks, organs and electronic devices with high speed and high spatial resolution.

Epigenetic regulation of the metallothionein-1A promoter by PU.1 during differentiation of THP-1 cells.

We recently demonstrated that metallothionein (MT)-1 A is a direct target gene negatively regulated by PU.1. In this study, we revealed that the expression of PU.1 was increased and accompanied by downregulation of MT-1A expression during TPA-induced THP-1 monocyte differentiation. Chromatin immunoprecipitation (ChIP) analysis demonstrated that PU.1 and the methyl CpG binding protein (MeCP) 2 bound to the same -887 to -602 region in the MT-1A promoter, and the binding of these proteins to this promoter was enhanced during differentiation. Consistently, bisulfite sequencing analyses around this region revealed that the proportion of methylated CpG sites was obivously increased during differentiation. In addition, ChIP analysis demonstrated that acetylated histone H4 around this region tended to be reduced and this may also play a role in the reduction of MT-1A expression during differentiation. Taken together, these findings suggest that MT-1A is epigenetically regulated by PU.1 during monocytic differentiation.

The differentiating and apoptotic effects of 2-aza-5'-deoxycytidine are dependent on the PU.1 expression level in PU.1-transgenic K562 cells.

The use of 5-aza-2'-deoxycytidine (5-azadc) for myelodysplastic syndrome, acute myeloid leukemia and chronic myeloid leukemia is becoming an effective and attractive option for these hematological malignancies. The PU.1 transcription factor is important for cellular differentiation through the control of its target genes not only in myeloid and B-lymphoid cells, but also in erythroid cells. Downregulation of PU.1 was reported to play a role in the pathogenesis of various hematological malignancies. In this study, we sought to identify the relationship between the effects of 5-azadc and PU.1. For this purpose, we employed PU.1-knockdown K562 (K562 PU.1KD) cells stably expressing PU.1 short inhibitory RNAs and PU.1-overexpressing K562 (K562 PU.1OE) cells. Therapeutic concentrations (0.1 and 0.5 µM) of 5-azadc resulted in growth arrest in the G2/M phase. Strikingly, however, K562 PU.1OE cells had significantly increased rates of G2/M and apoptotic sub-G1 phase cells. We observed the induction of cyclin B1, a regulator of the G2/M transition, after the addition of 5-azadc. This induction was abolished in K562 PU.1KD cells, but significantly induced in K562 PU.1OE cells. Further analyses revealed potent induction of hemoglobin A1 expression in K562 PU.1OE cells. Taken together, these findings suggest that the PU.1 expression level is tightly related to the differentiating and apoptotic effects of 5-azadc in K562 cells.

Transcription Factor c-Maf Promotes Immunoregulation of Programmed Cell Death 1-Expressed CD8+ T Cells in Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS. CD8+ T cells are prominently found at inflammatory sites. Recent advances in understanding checkpoint molecules, including programmed cell death 1 (PD-1), expressed on CD8+ T cells, highlight the immune regulatory roles of this T-cell subset; however, the role of CD8+ T cells in MS is unclear. Thus, we aimed to reveal the characteristics of PD-1-expressed (PD-1+) CD8+ T cells in MS. METHODS: We performed a cohort, case-control study for phenotyping analysis of PD-1+CD8+ T cells in disease remission and flare states using CSF and peripheral blood samples of 45 patients with MS or clinically isolated syndrome and 12 healthy subjects. We further analyzed the transcriptome of sorted PD-1+CD8+ T cells obtained from interferon (IFN)-ß-treated patients and validated their regulatory machinery using in vitro cell culture assays with lentiviral gene transfer. RESULTS: In the disease remission state, PD-1+CD8+ T cells were decreased in the peripheral blood of patients with MS and resolved in patients treated with IFN-ß treatment who showed immune regulatory cytokine interleukin (IL)-10 expression. In the disease flare state, we found that PD-1+CD8+ T cells were enriched in the CSF, which predicted a good response to subsequent IV steroid therapy. Transcriptome analysis of sorted PD-1+CD8+ T cells revealed the transcription factor c-Maf as a potential major regulator of the gene module, including multiple coinhibitory molecules. Furthermore, c-Maf expressed in CD8+ T cells induced PD-1 expression and production of IL-10 as well as suppressed alloactivated CD4+ T-cell survival. DISCUSSION: This study uncovered a favorable role of PD-1+CD8+ T cells against MS and demonstrated that c-Maf-driven IL-10 is an immune regulatory machinery.

Mitocryptide-2: purification, identification, and characterization of a novel cryptide that activates neutrophils.

Neutrophils are a class of leukocytes involved in innate immunity by monitoring and scavenging invading microorganisms and toxic substances. The actions of neutrophils in damaged tissues are still not well understood, particularly in the early stage of inflammation, and as-yet-unknown neutrophil-activating substances are proposed to induce their acute transmigration and activation. Here, we isolated and identified from porcine hearts a neutrophil-activating peptide. Structural analyses indicated that the primary structure of this peptide is formyl-Met-Thr-Asn-Ile-Arg-Lys-Ser-His-Pro-Leu-Met-Lys-Ile-Ile-Asn, which is identical to that of the N-terminal pentadecapeptide of porcine mitochondrial cytochrome b; we therefore named the newly isolated peptide "mitocryptide-2" (MCT-2), since we have recently purified and identified mitocryptide-1, a different class of a neutrophil-activating peptide. Synthetic MCT-2 and its human homolog hMCT-2 induced beta-hexosaminidase release in and chemotaxis of HL-60 cells differentiated into neutrophilic/granulocytic cells. The induction of beta-hexosaminidase release, chemotaxis, and the increase in the intracellular free Ca(2+) concentration by hMCT-2 were completely suppressed by pertussis toxin, indicating the involvement of G(i)- or G(o)-type G proteins in the signaling pathways. Moreover, MCT-2 and hMCT-2 also stimulated beta-hexosaminidase secretion in human neutrophils isolated from peripheral blood in a concentration-dependent manner. Additionally, these peptides partially competed with [(3)H]formyl-Met-Leu-Phe binding to HL-60 cells differentiated into neutrophilic/granulocytic cells, presenting the possibility that the receptor for MCT-2 and hMCT-2 is one of the formyl peptide receptors. These results demonstrate that MCT-2 and its human homolog hMCT-2 are cryptides that activate neutrophils, thus suggesting the presence of regulatory mechanisms involving such mitocryptides in innate immunity.

Experience Affects EEG Event-Related Synchronization in Dancers and Non-dancers While Listening to Preferred Music.

EEGs were analyzed to investigate the effect of experiences in listening to preferred music in dancers and non-dancers. Participants passively listened to instrumental music of their preferred genre for 2 min (Argentine tango for dancers, classical, or jazz for non-dancers), alternate genres, and silence. Both groups showed increased activity for their preferred music compared to non-preferred music in the gamma, beta, and alpha frequency bands. The results suggest all participants' conscious recognition of and affective responses to their familiar music (gamma), appreciation of the tempo embedded in their preferred music and emotional arousal (beta), and enhanced attention mechanism for cognitive operations such as memory retrieval (alpha). The observed alpha activity is considered in the framework of the alpha functional inhibition hypothesis, in that years of experience listening to their favorite type of music may have honed the cerebral responses to achieve efficient cortical processes. Analyses of the electroencephalogram (EEG) activity over 100s-long music pieces revealed a difference between dancers and non-dancers in the magnitude of an initial alpha event-related desynchronization (ERD) and the later development of an alpha event-related synchronization (ERS) for their preferred music. Dancers exhibited augmented alpha ERD, as well as augmented and uninterrupted alpha ERS over the remaining 80s. This augmentation in dancers is hypothesized to be derived from creative cognition or motor imagery operations developed through their dance experiences.

Speech and span: working memory capacity impacts the use of animacy but not of world knowledge during spoken sentence comprehension.

We present results from a study demonstrating that high- and low-span listeners show qualitatively different brain responses when comprehending simple active sentences. Participants listened to naturally produced sentences in three conditions in which the plausibility of thematic relations was manipulated, for instance: The dog(1)/The poet(2)/The box(3) is biting the mailman. Event-related potentials were recorded to the first noun, the verb, and the second noun in all three conditions. In (2), the thematic relations between the words in the sentence are less expected given our world knowledge, and this resulted in an N400 effect of semantic processing difficulty to the second noun for both high- and low-span subjects. In (3), the inanimate first noun cannot be the agent of the verb. Only high-span subjects showed an effect of animacy on the sentence-initial nouns, evident from a larger anterior negative shift to inanimate than animate nouns. Furthermore, to the thematically violated verbs (3), low-span subjects showed an N400, whereas high-span subjects generated a P600. We suggest that this P600 effect to the thematically violated verb may be related to processing costs resulting from a conflict between the provisional thematic roles assigned as a function of the inanimate sentence-initial noun, and the actual (animate) agent required by the verb. We further argue that low-span subjects lag behind those with high span in their use of animacy, but not real-world knowledge in the on-line computation of thematic roles in spoken language comprehension.

Repeated methamphetamine treatment impairs recognition memory through a failure of novelty-induced ERK1/2 activation in the prefrontal cortex of mice.

BACKGROUND: Recent clinical studies have suggested that chronic use of methamphetamine (METH) induces long-term cognitive deficits. To clarify the mechanism of METH-induced cognitive impairment, we investigated the effect of METH on cognitive function in mice. METHODS: Mice were repeatedly administered METH for 7 days, and their cognitive function was assessed using a novel-object recognition task. Therapeutic effects of clozapine and haloperidol on METH-induced cognitive impairment were investigated. Western blotting and specific inhibitors were employed to determine the role of extracellular signal-regulated kinase 1/2 (ERK1/2). RESULTS: Repeated METH treatment induced an impairment of recognition of novel objects and behavioral sensitization. These effects persisted for at least 28 days after the drug withdrawal. Clozapine, but not haloperidol, reduced METH-induced cognitive impairment. Hyperphosphorylation of ERK1/2 was found in the prefrontal cortex of mice exposed to the novel objects, but was abolished in mice treated with METH. Inhibition of ERK1/2 by the microinjection of PD98059 into the prefrontal cortex resulted in cognitive impairment. CONCLUSIONS: These results suggest that repeated METH treatment induces cognitive impairment, which is associated with the dysfunction of the ERK1/2 pathway in the prefrontal cortex.

The roles of sequencing and verbal working memory in sentence comprehension deficits in Parkinson's disease.

Studies of sentence comprehension deficits in Parkinson's disease (PD) patients suggest that language processing involves circuits connecting subcortical and cortical regions. Anatomically segregated neural circuits appear to support different cognitive and motor functions. To investigate which functions are implicated in PD comprehension deficits, we tested comprehension, verbal working memory span, and cognitive set-switching in a non-linguistic task in 41 PD patients; we also obtained speech measurements reflecting motor sequencing processes that may be involved in articulatory rehearsal within working memory. Comprehension of sentences with center-embedded or final relative clauses was impaired when they could not be understood from lexical semantic content alone. Overall comprehension error rates correlated strongly with impaired set-switching and significantly with reduced working memory span and speech motor sequencing deficits. Correlations with comprehension of different sentence structures indicate that these impairments do not represent a single deficit; rather, PD comprehension deficits appear to arise from several independent mechanisms. Deficits in cognitive set-switching or underlying inhibitory processes may compromise the ability to process relative clauses. Deficits in verbal working memory appear to impair comprehension of long-distance dependencies. Speech sequencing correlated with neither set-switching nor verbal working memory span, consistent with their being supported by independent, segregated cortico-subcortical circuits.

Neurodevelopmental outcome in children with posthemorrhagic hydrocephalus.

To determine the factors affecting the neurodevelopmental outcome in children with posthemorrhagic hydrocephalus, 78 children with intraventricular hemorrhage grade 3 or 4 were analyzed concerning the outcome in relation to the grade of intraventricular hemorrhage and intervention (surgical, medical, or no intervention) by means of a follow-up study. The mean age of the subjects at the last follow-up was 9.8 years. In children with intraventricular hemorrhage grade 4 with parenchymal hemorrhage, the outcomes in the group not requiring intervention were better than those in the groups requiring intervention, whereas in children with intraventricular hemorrhage grade 3 without parenchymal hemorrhage, there were no differences in the outcomes among the three groups with and without intervention. For the subjects who had undergone the same intervention, the outcomes in children with intraventricular hemorrhage grade 4 were worse than those in children with intraventricular hemorrhage grade 3. The outcomes in the children with surgical intervention only correlated with the grade of intraventricular hemorrhage. From these findings, we concluded that the outcomes in children with posthemorrhagic hydrocephalus were far more affected by the existence or extent of parenchymal hemorrhage than by the hydrocephalic process, which was suggested to be effectively controlled by the intervention.

Deficits in thematic integration processes in Broca's and Wernicke's aphasia.

This study investigated how normal subjects and Broca's and Wernicke's aphasics integrate thematic information incrementally using syntax, lexical-semantics, and pragmatics in a simple active declarative sentence. Three priming experiments were conducted using an auditory lexical decision task in which subjects made a lexical decision on a 'target' (the last word of each sentence) preceded by a 'prime' (a subject noun phrase and verb). The presence and magnitude of priming was compared to a baseline condition in which non-words systematically replaced real word primes. Normal subjects showed evidence for combinatorial thematics by exhibiting significant and larger amounts of thematic priming in the condition where two real words were present in the prime than in the conditions in which only one real word was present in the prime. Additionally, normal subjects showed sensitivity to both syntactic structures and pragmatics. In contrast, Broca's patients did not show significant priming for any condition nor did they show a difference in the magnitude of priming among the conditions. Nonetheless, they showed sensitivity to pragmatics. Wernicke's patients showed significant priming for all conditions, but did not show differences in the magnitude of priming among the conditions. However, they showed sensitivity to sentence grammaticality and pragmatics. The distinct patterns of performance of Broca's and Wernicke's aphasics are discussed in terms of the nature of their impairments in the processes of combinatorial thematics.

Establishment of a new canine cell line (CCT) originated from a cutaneous malignant histiocytosis.

A new canine cell line, named CCT, was established from the cutaneous malignant histiocytosis in a 4-year-old male Borzoi. CCT proliferated with loose adherence and doubling time was approximately 30 hr. When co-cultured with latex beads, CCT phagocytized beads vigorously. Lysozyme and vimentin were positive by immunostaining, and non-specific esterase and acid phosphatase were positive by cytochemical staining. These features indicated the cells had a histiocytic nature. Furthermore, by subcutaneous injection to nude mice CCT could successfully form tumors with the morphological and immunohistochemical features similar to the original tumor.

A PU.1 suppressive target gene, metallothionein 1G, inhibits retinoic acid-induced NB4 cell differentiation.

We recently revealed that myeloid master regulator SPI1/PU.1 directly represses metallothionein (MT) 1G through its epigenetic activity of PU.1, but the functions of MT1G in myeloid differentiation remain unknown. To clarify this, we established MT1G-overexpressing acute promyelocytic leukemia NB4 (NB4MTOE) cells, and investigated whether MT1G functionally contributes to all-trans retinoic acid (ATRA)-induced NB4 cell differentiation. Real-time PCR analyses demonstrated that the inductions of CD11b and CD11c and reductions in myeloperoxidase and c-myc by ATRA were significantly attenuated in NB4MTOE cells. Morphological examination revealed that the percentages of differentiated cells induced by ATRA were reduced in NB4MTOE cells. Since G1 arrest is a hallmark of ATRA-induced NB4 cell differentiation, we observed a decrease in G1 accumulation, as well as decreases in p21WAF1/CIP1 and cyclin D1 inductions, by ATRA in NB4MTOE cells. Nitroblue tetrazolium (NBT) reduction assays revealed that the proportions of NBT-positive cells were decreased in NB4MTOE cells in the presence of ATRA. Microarray analyses showed that the changes in expression of several myeloid differentiation-related genes (GATA2, azurocidin 1, pyrroline-5-carboxylate reductase 1, matrix metallopeptidase -8, S100 calcium-binding protein A12, neutrophil cytosolic factor 2 and oncostatin M) induced by ATRA were disturbed in NB4MTOE cells. Collectively, overexpression of MT1G inhibits the proper differentiation of myeloid cells.

The differentiation effect of low-dose cytosine arabinoside is disturbed in PU.1-knockdown K562 cells.

We recently demonstrated by using PU.1-knockdown K562 (K562 PU.1KD) cells stably expressing PU.1 short inhibitory RNAs and PU.1-overexpressing K562 (K562 PU.1OE) cells, that therapeutic concentrations of 5-aza-2'-deoxycytidine (5-azadC) induce erythroid differentiation of these cells and that the PU.1 expression level is closely associated with the differentiating and apoptotic effects of 5-azadC on K562 cells. In this study, we investigated whether the effects of low-dose cytosine arabinoside (Ara-C), which is another erythroid differentiation inducer in K562 cells, is associated with the expression level of PU.1 in these cells. As a result, we demonstrated that the effect of Ara-C on cell viability and differentiation, as determined by the WST-8 assay and ß-globin mRNA expression analysis, respectively, was suppressed in K562 PU.1KD cells compared to their controls. Collectively, these findings suggest that sufficient expression of PU.1 is indispensable for the erythroid differentiation of K562 cells.

Electrophysiological response to omitted stimulus in sentence processing.

The current study provides evidence that the absence of a syntactically expected item leads to a sustained cognitive processing demand. Event-related potentials were measured at the omission of a syntactically expected object argument in a speech sequence. English monolingual adults listened to paired sentences. The first sentence in the pair established a context. The second sentence provided a response to the first sentence that was either grammatically correct by containing an overt object argument in the form of a pronoun, or was syntactically unacceptable by omitting the expected object pronoun. Event-related potentials measured at the omission of the object argument showed a prolonged positivity for 100-600 ms with a broad scalp distribution, and for 600-1000 ms with a focus in the anterior region. This observed omitted stimulus potential may contain characteristics of the P300 component, associated with the detection of the deviation of an expected stimulus, and the classical P600 related to syntactic reanalysis. Further, the late anterior P600 may indicate an increased memory demand in sentence comprehension. Thus, this linguistic omitted stimulus potential is a cognitive indicator of language processing that can be used to investigate the organization of linguistic knowledge.

Isolation and identification of novel neutrophil-activating cryptides hidden in mitochondrial cytochrome C.

Although it is known that neutrophils infiltrate damaged sites immediately after tissue injury, the endogenous factors that induce their acute transmigration and activation have not been thoroughly investigated. For the candidates of those factors, we recently discovered two novel neutrophil-activating cryptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial proteins. In addition, many unknown neutrophil-activating peptides other than MCT-1 and MCT-2 were also observed during their purification. Here, we isolated and purified a novel neutrophil-activating peptide from porcine hearts, which we showed by structural analyses to have an identical primary structure to porcine mitochondrial cytochrome c (68-85). We named this novel functional octadecapeptide as mitocryptide-CYC (MCT-CYC). Structure-activity relationships of cytochrome c on ß-hexosaminidase (ß-HA) release from neutrophilic-differentiated HL- 60 cells demonstrated that peptides derived from the C-terminal part of cytochrome c induced ß-HA release and that cytochrome c (70-85) was the most potent cryptide among them. Since cytochrome c is known to be involved in the apoptotic process, our results suggest that cryptides, including MCT-CYC, derived from mitochondrial cytochrome c are possible factors that induce scavenging of toxic debris produced from apoptotic cells by neutrophils.

Queen succession through asexual reproduction in termites.

The evolution and maintenance of sexual reproduction may involve important tradeoffs because asexual reproduction can double an individual's contribution to the gene pool but reduces diversity. Moreover, in social insects the maintenance of genetic diversity among workers may be important for colony growth and survival. We identified a previously unknown termite breeding system in which both parthenogenesis and sexual reproduction are conditionally used. Queens produce their replacements asexually but use normal sexual reproduction to produce other colony members. These findings show how eusociality can lead to extraordinary reproductive systems and provide important insights into the advantages and disadvantages of sex.

Transient upregulation of indoleamine 2,3-dioxygenase in dendritic cells by human chorionic gonadotropin downregulates autoimmune diabetes.

OBJECTIVE: Pregnancy induces a state of immunological tolerance that aims at suppressing immune responses against the fetus and has been linked to temporal remission of preexisting autoimmune disorders. To understand the mechanisms of this reversible immune regulation, we investigated the role of a key pregnancy hormone, human chorionic gonadotropin (hCG), in immune tolerance against autoimmune type 1 diabetes in nonobese diabetic (NOD) mice. RESEARCH DESIGN AND METHODS: We injected hCG into cytokine gene-deficient NOD mice and evaluated the effects of hCG administration on T-cells and dendritic cells (DCs). RESULTS: We show that administration of hCG to NOD mice inhibits both the activation of diabetogenic CD4(+) and CD8(+) T-cells, in vitro and in vivo, and the progression of type 1 diabetes by upregulating the expression of indoleamine 2,3-dioxygenase (IDO) in DCs. IDO upregulation is transient and declined shortly after hCG withdrawal. DC depletion restores the diabetetogenic activity of splenic T-cells from hCG-treated mice, and inhibition of IDO activity by 1-methyl-tryptophan abrogates the hCG-induced T-cell suppression and resistance to type 1 diabetes. CONCLUSIONS: We propose that hCG-induced upregulation of IDO in DCs plays a major role in pregnancy-associated resistance to autoimmunity.

Characterization of gene expression profiles in early bovine pregnancy using a custom cDNA microarray.

Gene expression analysis comparing nonpregnant with pregnant bovine uteri, including placenta, was performed with a custom cDNA microarray containing 1,933 independent genes. These genes were classified into six categories according to biological function, as follows: cell and tissue structural dynamics (108 genes), intercellular communication (221), intracellular metabolism (265), cell cycle and apoptosis (26), regulation of gene expression (113), expressed sequence tag (EST) and function unknown (617), and uncomplemented genes (583 clones). This array possessed bovine placental/endometrial specificity, as it included many pregnancy-specific molecules, such as pregnancy-associated glycoprotein-1 (PAGs), placental lactogen (PLs), and prolactin-related protein-1 (PRPs). A total of 77 genes were induced and 12 repressed in the placenta/endometrium. Our results point to a fundamental role for bovine placental-specific genes such as PAGs, PLs, and PRPs, in implantation and placentogenesis, and document that cDNA microarray analysis from bovine placenta/endometrium is possible and is a specific tool for monitoring genome-wide gene expression during the establishment and maintenance of pregnancy.