Neural Substrates for Judgment of Self-Agency in Ambiguous Situations

The sense of agency is the attribution of oneself as the cause of one’s own actions and their effects. Accurate agency judgments are essential for adaptive behaviors in dynamic environments, especially in conditions of uncertainty. However, it is unclear how agency judgments are made in ambiguous situations where self-agency and non-self-agency are both possible. Agency attribution is thus thought to require higher-order neurocognitive processes that integrate several possibilities. Furthermore, neural activity specific to self-attribution, as compared with non-self-attribution, may reflect higher-order critical operations that contribute to constructions of self-consciousness. Based on these assumptions, the present study focused on agency judgments under ambiguous conditions and examined the neural correlates of this operation with functional magnetic resonance imaging. Participants performed a simple but demanding agency-judgment task, which required them to report on whether they attributed their own action as the cause of a visual stimulus change. The temporal discrepancy between the participant’s action and the visual events was adaptively set to be maximally ambiguous for each individual on a trial-by-trial basis. Comparison with results for a control condition revealed that the judgment of agency was associated with activity in lateral temporo-parietal areas, medial frontal areas, the dorsolateral prefrontal area, and frontal operculum/insula regions. However, most of these areas did not differentiate between self- and non-self-attribution. Instead, self-attribution was associated with activity in posterior midline areas, including the precuneus and posterior cingulate cortex. These results suggest that deliberate self-attribution of an external event is principally associated with activity in posterior midline structures, which is imperative for self-consciousness.     

New ellagitannin and galloyl esters of phenolic glycosides from sapwood of Quercus mongolica var. crispula (Japanese oak)

Two novel glycosides, 4,5-dimethoxy-3-hydroxyphenol 1-O-β-(6′-O-galloyl)-glucopyranoside (1) and (+)-2α-O-galloyl lyoniresinol 3α-O-β-d-xylopyranoside (2), as well as a novel ellagitannin named epiquisqualin B (3), were isolated from sapwood of Quercus mongolica var. crispula along with 19 known phenolic compounds. The structures of the novel compounds were elucidated on the basis of chemical and spectroscopic investigation. Compound 2 is the first example of a lignan galloyl ester, and 3 is the oxidation product of vescalagin, which is the major ellagitannin of this plant.     

Development of a System to Monitor Laryngeal Movement during Swallowing Using a Bend Sensor

Background

Swallowing dysfunction (also known as dysphagia), which results in a deterioration of nutritional intake, slows rehabilitation and causes aspiration pneumonia, is very common following neurological impairments. Although videofluorographic (VF) examination is widely used for detecting aspiration, an objective and non-invasive method for assessing swallowing function has yet to be established because of a lack of adequate devices and protocols. In this paper, a bend sensor whose resistance is altered by bending was introduced to monitor swallowing-related laryngeal movement.

Methods

Six healthy male volunteers were recruited in the present study. Specific time points on the signal waveform produced by the bend sensor were defined to describe laryngeal movement by differential analysis. Additionally, the physiological significance of the obtained waveform was confirmed by analyzing the sequential correlations between the signal waveform from the bend sensor and hyoid bone kinetics simultaneously recorded by VF.

Results

Seven time points were successfully defined on the signal waveform to reference laryngeal movement. Each time point was well correlated with certain VF events, with evidence of no significant time lags, and there were positive correlations between waveform time points and matched VF events. Furthermore, obvious similarities were noticed between the duration of each phase on the signal waveform and the duration of the matched hyoid bone activity.

Conclusions

The present monitoring system using a bend sensor might be useful for observing the temporal aspects of laryngeal movement during swallowing, and it was well coordinated with hyoid bone movement.     

Functions of beta2-adrenergic receptor in human periodontal ligament cells

Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, β2 adrenergic receptor (β2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through β2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that β2-AR expression in PDL tissues and their features in PDL cells. β2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high β2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, β2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing β2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific β2-AR agonist, fenoterol (FEN). Overexpression of β2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for β2-AR expression in PDL tissue and β2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through β2-AR might be important for restoration and homeostasis of PDL tissue.     

Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (Ret^M919T, Ret^N396K and Ret^Y792F) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. Ret^M919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in Ret^M919T/- mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.     

CXCL12 and TP53 genetic polymorphisms as markers of susceptibility in a Brazilian children population with acute lymphoblastic leukemia (ALL)

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Genetic polymorphisms in the 3′UTR region of the CXCL12 (rs1801157) and TP53 codon 72 (rs1042522) genes may contribute to susceptibility to childhood ALL because they affect some important processes, such as metastasis regulation and tumor suppression. Thus the objective of the present study was to detect the frequency of two genetic polymorphisms in ALL patients and controls and to add information their impact on genetic susceptibility and prognosis. The CXCL12 and TP53 polymorphisms were tested in 54 ALL child patients and in 58 controls by restriction fragment length polymerase chain reaction and allelic specific chain reaction techniques, respectively. The frequencies of both allelic variants were higher in ALL patients than in the controls and indicated a positive association: OR = 2.44; 95 % CI 1.05–5.64 for CXCL12 and OR = 2.20; 95 % CI 1.03–4.70 for TP53. Furthermore, when the two genetic variants were analyzed together, they increased significantly more than fivefold the risk of this neoplasia development (OR = 5.24; 95 % CI 1.39–19.75), indicating their potential as susceptibility markers for ALL disease and the relevance of the allelic variant combination to increased risk of developing malignant tumors. Future studies may indicate a larger panel of genes involved in susceptibility of childhood ALL and other hematological neoplasias.     

Hyperthermia inhibits homologous recombination repair and sensitizes cells to ionizing radiation in a time‐ and temperature‐dependent manner

Hyperthermia has long been known as a radio‐sensitizing agent that displays anti‐tumor effects, and has been developed as a therapeutic application. The mechanisms of hyperthermia‐induced radio‐sensitization are highly associated with inhibition of DNA repair. Our investigations aimed to show how hyperthermia inactivate homologous recombination repair in the process of sensitizing cells to ionizing radiation by using a series of DNA repair deficient Chinese Hamster cells. Significant differences in cellular toxicity attributable to hyperthermia at and above 42.5°C were observed. In wild‐type and non‐homologous end joining repair mutants, cells in late S phase showed double the amount heat‐induced radio‐sensitization effects of G1‐phase cells. Both radiation‐induced DNA double strand breaks and chromatin damage resulting from hyperthermia exposure was measured to be approximately two times higher in G2‐phase cells than G0/G1 cells. Additionally, G2‐phase cells took approximately two times as long to repair DNA damage over time than G0/G1‐phase cells. To supplement these findings, radiation‐induced Rad51 foci formations at DNA double strand break sites were observed to gradually dissociate in response to the temperature and time of hyperthermia exposure. Dissociated Rad51 proteins subsequently re‐formed foci at damage sites with time, and occurred in a trend also related to temperature and time of hyperthermia exposure. These findings suggest Rad51's dissociation and subsequent reformation at DNA double strand break sites in response to varying hyperthermia conditions plays an important role in hyperthermia‐induced radio‐sensitization. J. Cell. Physiol. 228: 1473–1481, 2013. © 2012 Wiley Periodicals, Inc.     

Optineurin suppression causes neuronal cell death via NF‐κB pathway

Mutations in more than 10 genes are reported to cause familial amyotrophic lateral sclerosis (ALS). Among these genes, optineurin (OPTN) is virtually the only gene that is considered to cause classical ALS by a loss‐of‐function mutation. Wild‐type optineurin (OPTN^WT) suppresses nuclear factor‐kappa B (NF‐κB) activity, but the ALS‐causing mutant OPTN is unable to suppress NF‐κB activity. Therefore, we knocked down OPTN in neuronal cells and examined the resulting NF‐κB activity and phenotype. First, we confirmed the loss of the endogenous OPTN expression after siRNA treatment and found that NF‐κB activity was increased in OPTN‐knockdown cells. Next, we found that OPTN knockdown caused neuronal cell death. Then, overexpression of OPTN^WT or OPTN^E50K with intact NF‐κB‐suppressive activity, but not overexpression of ALS‐related OPTN mutants, suppressed the neuronal death induced by OPTN knockdown. This neuronal cell death was inhibited by withaferin A, which selectively inhibits NF‐κB activation. Lastly, involvement of the mitochondrial proapoptotic pathway was suggested for neuronal death induced by OPTN knockdown. Taken together, these results indicate that inappropriate NF‐κB activation is the pathogenic mechanism underlying OPTN mutation‐related ALS.

     

Photoreceptor Proteins Initiate Microglial Activation via Toll-like Receptor 4 in Retinal Degeneration Mediated by All-trans-retinal

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by delayed clearance of all-trans-retinal from the retina, and in a retinitis pigmentosa mouse model with impaired retinal pigment epithelium (RPE) phagocytosis. Mouse microglia displayed RPE cytotoxicity and increased production of inflammatory chemokines/cytokines, Ccl2, Il1b, and Tnf, after coincubation with ligands that activate innate immunity. Notably, phagocytosis of photoreceptor proteins increased the activation of microglia/macrophages and RPE cells isolated from model mice as well as wild-type mice. The mRNA levels of Tlr2 and Tlr4, which can recognize proteins as their ligands, were elevated in mice with retinal degeneration. Bone marrow-derived macrophages from Tlr4-deficient mice did not increase Ccl2 after coincubation with photoreceptor proteins. Tlr4^−/−Abca4^−/−Rdh8^−/− mice displayed milder retinal degenerative phenotypes than Abca4^−/−Rdh8^−/− mice. Additionally, inactivation of microglia/macrophages by pharmacological approaches attenuated mouse retinal degeneration. This study demonstrates an important contribution of TLR4-mediated microglial activation by endogenous photoreceptor proteins in retinal inflammation that aggravates retinal cell death. This pathway is likely to represent an underlying common pathology in degenerative retinal disorders.     

Directed evolution of poly[(R)-3-hydroxybutyrate] depolymerase using cell surface display system: functional importance of asparagine at position 285

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZ_RpiT1) consists of three functional domains to effectively degrade solid PHB materials, and its catalytic domain catalyzes the ester bond cleavage of the substrate. We performed the directed evolution of PhaZ_RpiT1 targeted at the catalytic domain in combination with the cell surface display method to effectively screen for mutants with improved p-nitrophenyl butyrate (pNPC4) activity. Mutated PhaZ_RpiT1 genes generated by error-prone PCR were fused to the oprI gene to display them as fusion proteins on Escherichia coli cell surface. Some cells displaying the mutant enzymes showed a two- to fourfold increase in pNPC4 hydrolysis activity relative to cells displaying wild-type enzyme. These mutant genes were recombined by a staggered extension process and the recombined enzymes were displayed to result in a five- to eightfold higher pNPC4 hydrolysis activity than the wild type. To further evaluate the mutation effects, unfused and undisplayed enzymes were prepared and applied to the hydrolysis of p-nitrophenyl esters having different chain lengths (pNPCn; n?=?2–6) and PHB degradation. One specific second-generation mutant showed an approximately tenfold increase in maximum rate for pNPC3 hydrolysis, although its PHB degradation efficiency at 1 μg/mL of enzyme concentration was approximately 3.5-fold lower than that of the wild type. Gene analysis showed that N285D or N285Y mutations were found in six of the seven improved second-generation mutants, indicating that Asn285 probably participates in the regulation of substrate recognition and may be more favorable for PHB degradation process than other amino acid residues.