Brd activity regulates Müller glia-dependent retinal regeneration in zebrafish.

The zebrafish retina possesses tremendous regenerative potential. Müller glia underlie retinal regeneration through their ability to reprogram and generate multipotent neuronal progenitors that re-differentiate into lost neurons. Many factors required for Müller glia reprogramming and proliferation have been identified; however, we know little about the epigenetic and transcriptional regulation of these genes during regeneration. Here, we determined whether transcriptional regulation by members of the Bromodomain (Brd) family is required for Müller glia-dependent retinal regeneration. Our data demonstrate that three brd genes were expressed in Müller glia upon injury. brd2a and brd2b were expressed in all Müller glia and brd4 was expressed only in reprogramming Müller glia. Utilizing (+)-JQ1, a pharmacological inhibitor of Brd function, we demonstrate that transcriptional regulation by Brds plays a critical role in Müller glia reprogramming and regeneration. (+)-JQ1 treatment prevented cell cycle re-entry of Müller glia and the generation of neurogenic progenitors. Modulating the (+)-JQ1 exposure window, we identified the first 48 h post-injury as the time-period during which Müller glia reprogramming occurs. (+)-JQ1 treatments after 48 h post-injury had no effect on the re-differentiation of UV cones, indicating that Brd function is required only for Müller glia reprogramming and not subsequent specification/differentiation events. Brd inhibition also prevented the expression of reprogramming genes like ascl1a and lepb in Müller glia, but not effector genes like mmp9, nor did it affect microglial recruitment after injury. These results demonstrate that transcriptional regulation by Brds plays a critical role during Müller glia-dependent retinal regeneration in zebrafish.

High Temperature Deformation Characteristics of Al-Zn-Mg Alloy Modified with CaO-Added Mg.

Hot torsion tests were performed on an Al-Zn-Mg alloy modified with CaO-added Mg to investigate the effects of the Mg additive on the high temperature deformation characteristics. Effective stress- strain curves and processing maps were established from the experimental results under a range of deformation conditions. The fracture strain of the CaO-added Al-Zn-Mg alloy was higher than that of the Al-Zn-Mg alloy. The CaO-added Al-Zn-Mg alloy did not show an instability region in the processing map but the commercial Al-Zn-Mg alloy exhibited adiabatic shear bands at low temperatures and at a high strain rate. The results shown in this study were attributed to the reduction of the second phase by the addition of CaO-added Mg.

Evaluation of the Hot Workability of Commercially Pure Ti Using Hot Torsion Tests.

Optimum processing conditions were obtained by evaluating the hot working behavior of commercially pure Ti using hot torsion tests. Hot torsion tests were conducted at temperatures ranging from 800 °C-1000 °C and strain rates ranging from 0.1-10 s-1. The flow curves show that the peak stress increases as the temperature decreases and the strain rate increases. The optimum processing conditions were derived by comparing the processing and activation energy maps. The microstructure was characterized based on various regions of the processing map. The activation energy for plastic deformation was obtained using the constitutive equation. The activation energy differs depending on the constituent phases.

Fbxl19 recruitment to CpG islands is required for Rnf20-mediated H2B mono-ubiquitination.

Histone H2B lysine 120 mono-ubiquitination (H2Bub1) catalyzed by Rnf20 has been implicated in normal differentiation of embryonic stem (ES) and adult stem cells. However, it remains unknown how Rnf20 is recruited to its specific target chromosomal loci for the establishment of H2Bub1. Here, we reveal that Fbxl19, a CxxC domain-containing protein, promotes H2Bub1 at the promoters of CpG island-containing genes by interacting with Rnf20. We show that up-regulation of Fbxl19 increases the level of global H2Bub1 in mouse ES cells, while down-regulation of Fbxl19 reduces the level of H2Bub1. Our genome-wide target mapping unveils the preferential occupancy of Fbxl19 on CpG island-containing promoters, and we further discover that chromosomal binding of Fbxl19 is required for H2Bub1 of its targets. Moreover, we reveal that Fbxl19 is critical for proper differentiation of ES cells in collaboration with Rnf20. Altogether, our results demonstrate that Fbxl19 recruitment to CpG islands is required for Rnf20-mediated H2B mono-ubiquitination.

Yap1 is dispensable for self-renewal but required for proper differentiation of mouse embryonic stem (ES) cells.

Yap1 is a transcriptional co-activator of the Hippo pathway. The importance of Yap1 in early cell fate decision during embryogenesis has been well established, though its role in embryonic stem (ES) cells remains elusive. Here, we report that Yap1 plays crucial roles in normal differentiation rather than self-renewal of ES cells. Yap1-depleted ES cells maintain undifferentiated state with a typical colony morphology as well as robust alkaline phosphatase activity. These cells also retain comparable levels of the core pluripotent factors, such as Pou5f1 and Sox2, to the levels in wild-type ES cells without significant alteration of lineage-specific marker genes. Conversely, overexpression of Yap1 in ES cells promotes nuclear translocation of Yap1, resulting in disruption of self-renewal and triggering differentiation by up-regulating lineage-specific genes. Moreover, Yap1-deficient ES cells show impaired induction of lineage markers during differentiation. Collectively, our data demonstrate that Yap1 is a required factor for proper differentiation of mouse ES cells, while remaining dispensable for self-renewal.

Pore Characteristics of Lotus-Type Porous Cu-Fe and Cu-Cr Alloys Fabricated by Unidirectional Solidification.

Lotus-type porous Cu-Fe and Cu-Cr with long cylindrical pores was fabricated by centrifugal casting under hydrogen atmosphere and the effect of alloying elements on pore characteristics of lotus-type porous Cu was investigated. For the lotus type porous Cu-Fe alloy, the porosity slightly decreased and the average pore diameter slightly increased with increasing Fe content. For the lotus-type porous Cu-Cr alloy, the porosity sharply decreased and the average pore diameter drastically increased with an increase in the Cr content. From these results, it was found that the pore evolution and growth are affected by alloying element and this leads to the change in the pore characteristics of lotus-type porous Cu-Fe and Cu-Cr alloys.

Effect of the Spray Distance on the Properties of High Velocity Oxygen-Fuel (HVOF) Sprayed WC-12Co Coatings.

In this study influence of spray distance on the properties of WC-12Co coatings deposited by HVOF was investigated. WC-12Co coating was sprayed at spray distance of 300, 385 and 450 mm. From microstructure observation, it is confirmed that the porosity of coatings increases with increasing the spray distance. The X-ray diffraction patterns indicate that the coatings consist of pure WC, W, and Co as well as W2C and Co6W6C phases. The increase of the spray distance accelerated the decarburization of coatings. From micro hardness tests, it was found that the hardness and the fracture toughness decreased with increasing spray distance. These mechanical properties would be related with not only porosity but also the degree of decarburization.

Hot Deformation Behavior of Hot-Extruded AA7175 Through Hot Torsion Tests.

The hot deformation behavior of hot-extruded AA7175 was investigated with flow curves and processing maps through hot torsion tests. The flow curves and the deformed microstructures revealed that dynamic recrystallization (DRX) occurred in the hot-extruded AA7175 during hot working. The failure strain was highest at medium temperature. This was mainly influenced by the dynamic precipitation of fine rod-shaped MgZn2. The processing map determined the optimal deformation condition for the alloy during hot working.

Effect of Zn on Pore Characteristics in Lotus-Type Porous Cu.

The effect of Zn on pore characteristics in lotus-type porous Cu alloy was investigated. The lotustype porous Cu-Zn alloys were fabricated with Zn content from 0.01 to 0.1 at% by the centrifugal casting method. The results demonstrated that the porosity was rarely affected by Zn content. However, the average pore diameter and pore number density of the lotus type porous Cu-Zn alloys were significantly affected by the Zn content. The average pore diameter decreased as the Zn content increased up to 0.01 at%, and then increased as the Zn content increased up to 0.1 at%. In contrast, the variations in the pore number density of the lotus-type porous Cu-Zn alloys showed the reversed tendency with respect to that of the average pore diameter. The increase in heterogeneous nucleation sites for pores attributed to the decreased average pore diameter and the increased pore number density.

Occipitocervical inclination: new radiographic parameter of neutral occipitocervical position.

PURPOSE: To describe occipitocervical inclination (OCI), a new parameter that could compensate for defects in existing radiographic parameters, and to define occipitocervical neutral position. METHODS: Neutral, flexion, and extension lateral cervical spine radiographs of 200 patients (100 male and 100 female patients) judged to be normal were analyzed. The mean age was 45.19 years (range 11-74; 42.84 for male and 47.53 for female patients). For OCI, the angle formed by the line connecting the posterior border of the C4 vertebral body and McGregor's line was measured. Occipitocervical angle (OCA) and occipitocervical distance (OCD) were measured and compared with OCI. RESULTS: OCI on standard, neutral lateral cervical radiographs was 102.51° ± 8.87°. There was no significant gender difference in neutral OCI 102.81° ± 7.93° for male and 102.21° ± 9.74° for female patients (P = 0.631). The mean neutral OCA was 38.69° ± 9.23°, and the mean neutral OCD was 22.98 ± 5.10 mm. Pearson's correlation coefficient for the value of the cervical lordosis angle and that of neutral OCI was r = 0.274 (P < 0.001). Intraclass correlation coefficient values for inter- and intraobserver reliability for OCI were significantly higher than those for OCA (P < 0.001) and tended to be higher than those for OCD (P = 0.087). CONCLUSIONS: OCI is a very useful parameter for the determination of neutral position during occipitocervical fusion for patients with altered C0-C2 anatomy.

Bcl6a function is required during optic cup formation to prevent p53-dependent apoptosis and colobomata.

Mutations in BCOR (Bcl6 corepressor) are found in patients with oculo-facio-cardio-dental (OFCD) syndrome, a congenital disorder affecting visual system development, and loss-of-function studies in zebrafish and Xenopus demonstrate a role for Bcor during normal optic cup development in preventing colobomata. The mechanism whereby BCOR functions during eye development to prevent colobomata is not known, but in other contexts it serves as a transcriptional corepressor that potentiates transcriptional repression by B cell leukemia/lymphoma 6 (BCL6). Here, we have explored the function of the zebrafish ortholog of Bcl6, Bcl6a, during eye development, and our results demonstrate that Bcl6a, like Bcor, is required to prevent colobomata during optic cup formation. Our data demonstrate that Bcl6a acts downstream of Vax1 and Vax2, known regulators of ventral optic cup formation and choroid fissure closure, and that bcl6a is a direct target of Vax2. Together, this regulatory network functions to repress p53 expression and thereby suppress apoptosis in the developing optic cup. Furthermore, our data demonstrate that Bcl6a functions cooperatively with Bcor, Rnf2 and Hdac1 in a common gene regulatory network that acts to repress p53 and prevent colobomata. Together, these data support a model in which p53-dependent apoptosis needs to be tightly regulated for normal optic cup formation and that Bcl6a, Bcor, Rnf2 and Hdac1 activities mediate this regulation.

Deep Neural Network-based Empirical Mode Decomposition for Motor Imagery EEG Classification.

Motor imagery refers to the brain's response during the mental simulation of physical activities, which can be detected through electroencephalogram (EEG) signals. However, EEG signals exhibit a low signal-to-noise ratio (SNR) due to various artifacts originating from other physiological sources. To enhance the classification performance of motor imagery tasks by increasing the SNR of EEG signals, several signal decomposition approaches have been proposed. Empirical mode decomposition (EMD) has shown promising results in extracting EEG components associated with motor imagery tasks more effectively than traditional linear decomposition algorithms such as Fourier and wavelet methods. Nevertheless, the EMD-based algorithm suffers from a significant challenge known as mode mixing, where frequency components intertwine with the intrinsic mode functions obtained through EMD. This issue severely hampers the accuracy of motor imagery classification. Despite numerous algorithms proposed, mode mixing remains a persistent issue. In this paper, we propose the Deep-EMD algorithm, a deep neural network-based approach to mode mixing problem. We employ two datasets to compare the motor imagery classification and mode mixing improvement achieved by the conventional EMD algorithm. Our experimental results demonstrate that the Deep-EMD algorithm effectively mitigates the mode mixing problem in decomposed EEG components, leading to improved motor imagery classification performance.

The active von Frey filament test: A new technique to evaluate hand tactile sensation in continuous variable units.

The von Frey filament test (vF) is a mainstay of clinical examination. However, its results can be affected by touch speed and other potentially confounding factors. Moreover, the differences between two adjacent filament levels are too large to detect subtle changes. Active vF (AvF) was developed to induce in-depth sensory change. The present study hypothesized that AvF produces different patterns of fingertip sensation; consequently, it could be used as a new assessment tool for neural impairment. The aim of the study was to provide preliminary normative comparative vF and AvF data. This study prospectively examined 32 healthy participants, using AvF and vF. The index and the fifth finger volar pad were examined using AvF and vF, without visual stimulation. The correlation between AvF and vF measurements was evaluated. In addition, differences according to innervation zone, right versus left hand, and gender, and the correlation between AvF values and subjects' age were analyzed. Mean AvF value was significantly higher and had greater variance than vF (111.3 ± 46.9 vs. 24.1 ± 9.8; P < 0.01). The Spearman correlation coefficient between AvF and vF was 0.341. Values were similar in the index and fifth fingers and right and left hands. However, values were significantly different between women and men. The correlation between age and AvF values was 0.259. AvF provided more precise values, with continuous units for tactile sensation, excluding tester-dependent factors. Furthermore, AvF and vF values may not be correlated.

Regulation of FOXO1 by TAK1-Nemo-like kinase pathway.

The FOXO family of forkhead transcription factors has a variety of important functions in stress response, metabolism, cell cycle, apoptosis, longevity, etc. The transcriptional activity and subcellular localization of FOXO are tightly regulated by post-translational modifications, including phosphorylation by various kinases. Here, we report that the transforming growth factor-beta-activated kinase (TAK1)-Nemo-like kinase (NLK) pathway negatively regulates FOXO1. We show that NLK binds and phosphorylates FOXO1 at Pro-directed Ser/Thr residues in the transactivation domain. The phosphorylation by TAK1-NLK pathway inhibits the transcriptional activity of FOXO1 and excludes FOXO1 from the nucleus, which is independent of phosphatidylinositol 3-kinase/Akt pathway. Consistently, knockdown of TAK1-NLK pathway dephosphorylates FOXO1 and decreases phospho-Ser-329 FOXO1 level. It also induces translocation of FOXO1 into the nucleus and leads to an increase in mRNA levels of FOXO target genes and poly(ADP-ribose) polymerase cleavage. In addition, we show the interaction between NLK and FOXO1 is evolutionarily conserved in Drosophila. Collectively, these findings provide the first evidence that TAK1-NLK pathway is a novel regulator of FOXO1.

Potential Novel Genes for Late-Onset Alzheimer's Disease in East-Asian Descent Identified by APOE-Stratified Genome-Wide Association Study.

The present study reports two novel genome-wide significant loci for late-onset Alzheimer's disease (LOAD) identified from APOE ε4 non-carrier subjects of East Asian origin. A genome-wide association study of Alzheimer's disease was performed in 2,291 Korean seniors in the discovery phase, from the Gwangju Alzheimer' and Related Dementias (GARD) cohort study. The study was replicated in a Japanese cohort of 1,956 subjects that suggested two novel susceptible SNPs in two genes: LRIG1 and CACNA1A. This study demonstrates that the discovery of AD-associated variants is feasible in non-European ethnic groups using samples comprising fewer subjects from the more homogeneous genetic background.

Analysis of the Change Patterns of Sagittal Alignment Values After Selective Thoracic Fusion in Lenke 1 Adolescent Idiopathic Scoliosis According to Preoperative Thoracic Kyphosis Status.

STUDY DESIGN: This was a retrospective study. OBJECTIVE: The objective of this study was to demonstrate the different change patterns in reciprocal sagittal alignment values after selective thoracic fusion (STF) in Lenke type 1 adolescent idiopathic scoliosis (AIS) according to preoperative thoracic kyphosis (TK). SUMMARY OF BACKGROUND DATA: Several studies have found significant increase in TK after STF, while other studies have reported decrease in TK postoperatively. Similar inconclusive results on changes in lumbar lordosis (LL) have been reported, showing LL increase, decrease, or no change. MATERIALS AND METHODS: Ninety-three patients presenting with Lenke type 1 AIS treated by posterior STF with a minimum follow-up of 2 years were included in this study. Using whole spine radiographs, sagittal parameters including TK, LL, and upper lumbar lordosis (ULL) were compared preoperatively and at the last follow-up between a hypokyphosis group (preoperative TK<20 degrees) and a normokyphosis group (preoperative TK≥20 degrees). Health-related quality of life (HRQOL) was assessed using scoliosis research society health-related quality of life-30 (SRS-30) and short from health survey-36 questionnaire at the last visit. RESULTS: The mean follow-up duration was 74.9 months. In the hypokyphosis group (35 patients), TK, LL, and ULL statistically significantly increased after surgery by mean 7.7, 5.1, and 3.7 degrees (P<0.001, <0.001, and 0.001). In the normokyphosis group (58 patients), these parameters did not show significant changes after STF. Final TK was significantly lower in hypokyphosis group than that in the normokyphosis group (21.2 vs. 30.9 degrees, P<0.001) while final LL did not differ between 2 groups (52.4 vs. 54.6 degrees, P=0.194). HRQOL did not differ significantly between the 2 groups. CONCLUSIONS: After STF in Lenke 1 AIS, TK, and LL statistically significantly increased through an increase in the mean ULL in the hypokyphosis group while those mean values did not change in the normokyphosis group. Despite the final mean value of the TK in the hypokyphosis group increasing by 7.7 degrees, it was statistically significantly lower than the final mean TK value in the normokyphosis group which did not increase after STF surgery by posterior approach. However, HRQOL showed no significant difference between the 2 groups.

Zebrafish blowout provides genetic evidence for Patched1-mediated negative regulation of Hedgehog signaling within the proximal optic vesicle of the vertebrate eye.

In this study, we have characterized the ocular defects in the recessive zebrafish mutant blowout that presents with a variably penetrant coloboma phenotype. blowout mutants develop unilateral or bilateral colobomas and as a result, the retina and retinal pigmented epithelium are not contained within the optic cup. Colobomas result from defects in optic stalk morphogenesis whereby the optic stalk extends into the retina and impedes the lateral edges of the choroid fissure from meeting and fusing. The expression domain of the proximal optic vesicle marker pax2a is expanded in blowout at the expense of the distal optic vesicle marker pax6, suggesting that the initial patterning of the optic vesicle into proximal and distal territories is disrupted in blowout. Later aspects of distal optic cup formation (i.e. retina development) are normal in blowout mutants, however. Positional cloning of blowout identified a nonsense mutation in patched1, a negative regulator of the Hedgehog pathway, as the underlying cause of the blowout phenotype. Expanded domains of expression of the Hedgehog target genes patched1 and patched2 were observed in blowout, consistent with a loss of Patched1 function and upregulation of Hedgehog pathway activity. Moreover, colobomas in blowout could be suppressed by pharmacologically inhibiting the Hedgehog pathway with cyclopamine, and maximal rescue occurred when embryos were exposed to cyclopamine between 5.5 and 13 hours post-fertilization. These observations highlight the critical role that Hedgehog pathway activity plays in mediating patterning of the proximal/distal axis of the optic vesicle during the early phases of eye development and they provide genetic confirmation for the integral role that patched1-mediated negative regulation of Hedgehog signaling plays during vertebrate eye development.

Super-enhancer-guided mapping of regulatory networks controlling mouse trophoblast stem cells.

Trophectoderm (TE) lineage development is pivotal for proper implantation, placentation, and healthy pregnancy. However, only a few TE-specific transcription factors (TFs) have been systematically characterized, hindering our understanding of the process. To elucidate regulatory mechanisms underlying TE development, here we map super-enhancers (SEs) in trophoblast stem cells (TSCs) as a model. We find both prominent TE-specific master TFs (Cdx2, Gata3, and Tead4), and >150 TFs that had not been previously implicated in TE lineage, that are SE-associated. Mapping targets of 27 SE-predicted TFs reveals a highly intertwined transcriptional regulatory circuitry. Intriguingly, SE-predicted TFs show 4 distinct expression patterns with dynamic alterations of their targets during TSC differentiation. Furthermore, depletion of a subset of TFs results in dysregulation of the markers for specialized cell types in placenta, suggesting a role during TE differentiation. Collectively, we characterize an expanded TE-specific regulatory network, providing a framework for understanding TE lineage development and placentation.

Laminin beta1 and gamma1 containing laminins are essential for basement membrane integrity in the zebrafish eye.

PURPOSE: In this study recessive zebrafish mutations in the genes encoding laminin beta1 (lamb1) and laminin gamma1 (lamc1) were used to determine the functions of these laminin proteins during ocular basement membrane formation and during zebrafish eye development. METHODS: Ocular defects in lamb1 and lamc1 mutants were characterized by using a combination of histology, immunohistochemistry, in situ hybridization, and transmission electron microscopy. RESULTS: The results demonstrated that zebrafish lamb1 and lamc1 mutants possess defects in two ocular basement membranes--the lens capsule and the inner limiting membrane--whereas Bruch's membrane is largely unaffected. lamb1 and lamc1 mutants possess severe lens dysplasias that result from a compromise in lens capsule integrity. Inner limiting membrane continuity is irregular in these mutants, and these irregularities result in small retinal ectopias that extend from the retina into the interstitial space between the retina and the lens. At late embryonic stages (e.g., 5-7 days after fertilization), retinal lamination defects are also observed in a subset of laminin mutants. CONCLUSIONS: The results demonstrate that laminin beta1 and -gamma1 containing laminins are essential for the integrity of the lens capsule basement membrane and inner limiting membrane in the zebrafish eye.

BP75, bromodomain-containing M(r) 75,000 protein, binds dishevelled-1 and enhances Wnt signaling by inactivating glycogen synthase kinase-3 beta.

To identify novel regulators of Wnt signaling, we performed yeast two-hybrid analyses with Dvl-1 and identified BP75 as a candidate. Here, we demonstrated that BP75 directly interacts with Dvl-1 in mammalian cells and enhances TCF-dependent gene expression induced by Dvl-1. In support of these results, BP75 in cooperation with Dvl-1 was found to facilitate dephosphorylation at Tyr216 of glycogen synthase kinase-3beta and consequently inhibit its kinase activity. Furthermore, the nuclear translocation and formation of vesicular structures of beta-catenin were induced by BP75 and Dvl-1 in a synergistic manner. Collectively, these results provided us a novel mechanism in Wnt signaling where BP75 plays important regulatory roles between glycogen synthase kinase-3beta and Dvl.