Repurposing of the enhancer-promoter communication underlies the compensation of Mesp2 by Mesp1.

Organisms are inherently equipped with buffering systems against genetic perturbations. Genetic compensation, the compensatory response by upregulating another gene or genes, is one such buffering mechanism. Recently, a well-conserved compensatory mechanism was proposed: transcriptional adaptation of homologs under the nonsense-mediated mRNA decay pathways. However, this model cannot explain the onset of all compensatory events. We report a novel genetic compensation mechanism operating over the Mesp gene locus. Mesp1 and Mesp2 are paralogs located adjacently in the genome. Mesp2 loss is partially rescued by Mesp1 upregulation in the presomitic mesoderm (PSM). Using a cultured PSM induction system, we reproduced the compensatory response in vitro and found that the Mesp2-enhancer is required to promote Mesp1. We revealed that the Mesp2-enhancer directly interacts with the Mesp1 promoter, thereby upregulating Mesp1 expression upon the loss of Mesp2. Of note, this interaction is established by genomic arrangement upon PSM development independently of Mesp2 disruption. We propose that the repurposing of this established enhancer-promoter communication is the mechanism underlying this compensatory response for the upregulation of the adjacent gene.

The RNA helicase DDX6 controls early mouse embryogenesis by repressing aberrant inhibition of BMP signaling through miRNA-mediated gene silencing.

The evolutionarily conserved RNA helicase DDX6 is a central player in post-transcriptional regulation, but its role during embryogenesis remains elusive. We here show that DDX6 enables proper cell lineage specification from pluripotent cells by analyzing Ddx6 knockout (KO) mouse embryos and employing an in vitro epiblast-like cell (EpiLC) induction system. Our study unveils that DDX6 is an important BMP signaling regulator. Deletion of Ddx6 causes the aberrant upregulation of the negative regulators of BMP signaling, which is accompanied by enhanced expression of Nodal and related genes. Ddx6 KO pluripotent cells acquire higher pluripotency with a strong inclination toward neural lineage commitment. During gastrulation, abnormally expanded Nodal and Eomes expression in the primitive streak likely promotes endoderm cell fate specification while inhibiting mesoderm differentiation. We also genetically dissected major DDX6 pathways by generating Dgcr8, Dcp2, and Eif4enif1 KO models in addition to Ddx6 KO. We found that the miRNA pathway mutant Dgcr8 KO phenocopies Ddx6 KO, indicating that DDX6 mostly works along with the miRNA pathway during early development, whereas its P-body-related functions are dispensable. Therefore, we conclude that DDX6 prevents aberrant upregulation of BMP signaling inhibitors by participating in miRNA-mediated gene silencing processes. Overall, this study delineates how DDX6 affects the development of the three primary germ layers during early mouse embryogenesis and the underlying mechanism of DDX6 function.

P-body dynamics revealed by DDX6 protein knockdown via the auxin-inducible degron system.

The transcriptome dynamically changes via several transcriptional and post-transcriptional mechanisms. RNA-binding proteins contribute to such mechanisms to regulate the cellular status. DDX6 is one such protein and a core component of processing bodies (P-bodies), membrane-less cytosolic substructures where RNA and proteins localize and are functionally regulated. Despite the importance of DDX6, owing to the lack of tightly controlled methods for protein knockdown, it was difficult to assess in high time resolution how its depletion exactly affects the P-body assembly structure. Therefore, we adopted an advanced protein degradation method, the auxin-induced degron (AID) system, to degrade DDX6 acutely in ES cells. By introducing AID-tagged DDX6 and the E3 ligase subunit of OsTIR1 into ES cells, we successfully degraded DDX6 following auxin analog (indole-3-acetic acid, IAA) treatment. The degradation rate of DDX6 was slower than that of the cytosolic reporter protein EGFP but was enhanced by increasing the OsTIR1 dosage. Lastly, we confirmed that a substantial portion of P-bodies disappears around the time of 1 hr after IAA addition consistent with DDX6 depletion detected by western blot. In accordance with this, we detected transcriptome changes by 6 hr after IAA treatment. Therefore, we demonstrated the applicability of the AID method to gain insight into the function of P-bodies and their protein components.

Phosphorylation states change Otx2 activity for cell proliferation and patterning in the Xenopus embryo.

The homeodomain transcription factor Otx2 has essential roles in head and eye formation via the negative and positive regulation of its target genes, but it remains elusive how this dual activity of Otx2 affects cellular functions. In the current study, we first demonstrated that both exogenous and endogenous Otx2 are phosphorylated at multiple sites. Using Xenopus embryos, we identified three possible cyclin-dependent kinase (Cdk) sites and one Akt site, and analyzed the biological activities of phosphomimetic (4E) and nonphosphorylatable (4A) mutants for those sites. In the neuroectoderm, the 4E but not the 4A mutant downregulated the Cdk inhibitor gene p27xic1 (cdknx) and posterior genes, and promoted cell proliferation, possibly forming a positive-feedback loop consisting of Cdk, Otx2 and p27xic1 for cell proliferation, together with anteriorization. Conversely, the 4A mutant functioned as an activator on its own and upregulated the expression of eye marker genes, resulting in enlarged eyes. Consistent with these results, the interaction of Otx2 with the corepressor Tle1 is suggested to be phosphorylation dependent. These data suggest that Otx2 orchestrates cell proliferation, anteroposterior patterning and eye formation via its phosphorylation state.

Tissue substructure-specific deposition of the ß3-containing laminin-332 in the biliary epithelium of human and mouse livers.

Laminin is a family of basement membrane proteins, whose selective and spatiotemporal expression profiles are linked to their various functions in development, maintenance, and functional regulation of different tissues. In the liver, α1-and α5-containing laminin isoforms have been documented to be critically involved in the developmental process of the epithelial tissue of the bile duct. However, possible roles of other laminin isoforms in bile duct formation and function remain elusive. Here, we evaluated public single-cell RNA sequencing databases on human liver cells to reveal expression landscape of laminin genes, and found that genes for laminin-332 subunits were conjointly expressed in the EPCAM+ biliary epithelial cell population. Expression of the ß3 and γ2 subunit genes was restricted to biliary epithelial cells in the liver and, remarkably, showed apparent heterogeneity among them. We confirmed the heterogeneous nature of the laminin-ß3 expression in murine livers, which was firmly related to morphological substructures in the biliary epithelium. Finally, we generated the liver epithelial tissue-specific laminin- ß3 knockout mice and found that this laminin subunit was dispensable under physiological conditions. Together, our present findings have identified the ß3 subunit and the related laminin-332 isoform as useful markers and potentially important regulatory molecules for future understanding of pathophysiology in the hepatobiliary system.

The transcription factor Klf5 is essential for intrahepatic biliary epithelial tissue remodeling after cholestatic liver injury.

Under various conditions of liver injury, the intrahepatic biliary epithelium undergoes dynamic tissue expansion and remodeling, a process known as ductular reaction. Mouse models defective in inducing such a tissue-remodeling process are more susceptible to liver injury, suggesting a crucial role of this process in liver regeneration. However, the molecular mechanisms regulating the biliary epithelial cell (BEC) dynamics in the ductular reaction remain largely unclear. Here, we demonstrate that the transcription factor Krüppel-like factor 5 (Klf5) is highly enriched in mouse liver BECs and plays a key role in regulating the ductular reaction, specifically under cholestatic injury conditions. Although mice lacking Klf5 in the entire liver epithelium, including both hepatocytes and BECs (Klf5-LKO (liver epithelial-specific knockout) mice), did not exhibit any apparent phenotype in the hepatobiliary system under normal conditions, they exhibited significant defects in biliary epithelial tissue remodeling upon 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced cholangitis, concomitantly with exacerbated cholestasis and reduced survival rate. In contrast, mice lacking Klf5 solely in hepatocytes did not exhibit any such phenotypes, confirming Klf5's specific role in BECs. RNA-sequencing analyses of BECs isolated from the Klf5-LKO mouse livers revealed that the Klf5 deficiency primarily affected expression of cell cycle-related genes. Moreover, immunostaining analysis with the proliferation marker Ki67 disclosed that the Klf5-LKO mice had significantly reduced BEC proliferation levels upon injury. These results indicate that Klf5 plays a critical role in the ductular reaction and biliary epithelial tissue expansion and remodeling by inducing BEC proliferation and thereby contributing to liver regeneration.

Sub-picosecond regenerative amplifier of Yb-doped Y(2)O(3) ceramic thin disk.

We have developed a 1 kHz regenerative amplifier using an Yb:Y(2)O(3) ceramic thin disk as the gain medium. Furthermore, the thermal conductivity and heat generation property of Yb:Y(2)O(3) ceramic were investigated. In the developed regenerative amplifier, a laser beam is bounced off the thin disk six times in each round trip. The output energy is over 2 mJ, spectral bandwidth is 1.8 nm at FWHM, and pulse duration after pulse compression is 0.9 ps.

Polarization-selectable cavity locking method for generation of laser Compton scattered γ-rays.

Nowadays, generation of energy-tunable, monochromatic γ-rays is needed to establish a nondestructive assay method of nuclear fuel materials. The γ-rays are generated by collision of laser photons stored in a cavity and relativistic electrons. We propose a configuration of an enhancement cavity capable of performing polarization control fabricated by a combination of a four-mirror ring cavity with a small spot inside a cavity and a three-mirror of reflective optics as an image inverter for polarization-selectable γ-rays. The image inverter introduces a phase shift of specific polarization which can be used to generate an error signal to lock an optical cavity at a resonance condition.

Efficient generation of Xe K-shell x rays by high-contrast interaction with submicrometer clusters.

The interaction between a 25 TW laser and Xe clusters at a peak intensity of 1 × 10¹9 W/cm² has been investigated. Xe K-shell x rays, whose energies are approximately 30 keV, were clearly observed with a hard x-ray CCD at 3.4 MPa. Moreover, we studied the yield of the Xe K-shell x rays by changing the pulse duration of the laser at a constant laser energy and found that the pulse duration of 40 fs is better than that of 300 fs for generating Xe K-shell x rays.

The Roles of Dominance of the Nitric Oxide Fractions Nitrate and Nitrite in the Epilepsy-Prone EL Mouse Brain.

BACKGROUND: Oxidative stress is thought to be closely related to epileptogenesis. We have previously reported that nitric oxide (NO) levels are higher in epilepsy-prone EL mice between the ages of 3 and 8 weeks than in control mice. However, NO is divided into two fractions, nitrite (NO2) and nitrate (NO3), which appear to play different roles in epileptogenesis. METHODS: NO2 and NO3 levels were measured, in EL mice and the control mice, in the parietal cortex, which is thought to be the primary epileptogenetic center in EL mice, and measured in the hippocampus, which is thought to be the secondary center. RESULTS: NO3 levels in the hippocampus and parietal cortex of the immature EL mice (3 to 8 weeks of age) were significantly higher than those in the control mice; NO2 levels were significantly higher in the EL mice throughout the study period. The NO3 levels were significantly higher than the NO2 levels in the immature EL mice, but after the onset of ictogenesis at 10 weeks of age, the relative levels of the two fractions reversed. CONCLUSION: The reversal of the NO fraction distribution at the onset of seizures that we observed may be related to the developmental process of seizure susceptibility in the neural network of EL mice.

Laser ceramic materials for subpicosecond solid-state lasers using Nd3+-doped mixed scandium garnets.

We have successfully developed and demonstrated broadband emission Nd-doped mixed scandium garnets based on laser ceramic technology. The inhomogeneous broadening of Nd(3+) fluorescence lines results in a bandwidth above 5 nm that is significantly broader than that for Nd:YAG and enables subpicosecond mode-locked pulse durations. We have also found the emission cross section of 7.8 × 10(-20) cm(2) to be adequate for efficient energy extraction and thermal conductivity of 4.7 W/mK from these new Nd-doped laser ceramics. The new laser ceramics are good candidates for laser host material in a diode-pumped subpicosecond laser system with high efficiency and high repetition rate.

Photoreversible DNA end capping for the formation of hairpin structures.

We describe a photoreversible DNA end capping via 3-cyanovinylcarbazole nucleoside. Doubly end-capped oligodeoxynucleotide (ODN) exhibits increased stability against snake venom phosphodiesterase and shows high thermal stability.

High-spatiotemporal-quality petawatt-class laser system.

We have developed a femtosecond high-intensity laser system that combines both Ti:sapphire chirped-pulse amplification (CPA) and optical parametric CPA (OPCPA) techniques and produces more than 30 J broadband output energy, indicating the potential for achieving peak powers in excess of 500 TW. With a cleaned high-energy seeded OPCPA preamplifier as a front end in the system, for the compressed pulse without pumping the final amplifier, we found that the temporal contrast in this system exceeds 10(10) on the subnanosecond time scales, and is near 10(12) on the nanosecond time scale prior to the peak of the main femtosecond pulse. Using diffractive optical elements for beam homogenization of a 100 J level high-energy Nd:glass green pump laser in a Ti:sapphire final amplifier, we have successfully generated broadband high-energy output with a near-perfect top-hat-like intensity distribution.

Bacterial Meningitis Due to Streptococcus pneumoniae in a 7-Month-Old Girl Who Received Three Doses of 13-Valent Pneumococcal Conjugate Vaccine.

In Japan, pneumococcal vaccine has been routinely administered since 2010 to prevent invasive pneumococcal diseases such as Streptococcus pneumoniae meningitis. We describe a case of pneumococcal meningitis in a 7-month-old girl who had received three doses of 13-valent pneumococcal conjugate vaccine. Brain magnetic resonance imaging showed infarcts in the right frontal region, and she was treated with antibiotics, intravenous immunoglobulin, dexamethasone, and edaravone. On day 27, an enhanced brain CT scan showed improvement of abnormal findings in the frontal region, except for slight atrophy. The S. pneumoniae serotype was 12F, which is not included in the 13-valent pneumococcal conjugate vaccine. A future vaccine is expected to use cross-reactivity to target common antigens.

SNP genotyping by DNA photoligation: application to SNP detection of genes from food crops.

We describe a simple and inexpensive single-nucleotide polymorphism (SNP) typing method, using DNA photoligation with 5-carboxyvinyl-2'-deoxyuridine and two fluorophores. This SNP-typing method facilitates qualitative determination of genes from indica and japonica rice, and showed a high degree of single nucleotide specificity up to 10 000. This method can be used in the SNP typing of actual genomic DNA samples from food crops.

Heterogeneity and stochastic growth regulation of biliary epithelial cells dictate dynamic epithelial tissue remodeling.

Dynamic remodeling of the intrahepatic biliary epithelial tissue plays key roles in liver regeneration, yet the cellular basis for this process remains unclear. We took an unbiased approach based on in vivo clonal labeling and tracking of biliary epithelial cells in the three-dimensional landscape, in combination with mathematical simulation, to understand their mode of proliferation in a mouse liver injury model where the nascent biliary structure formed in a tissue-intrinsic manner. An apparent heterogeneity among biliary epithelial cells was observed: whereas most of the responders that entered the cell cycle upon injury exhibited a limited and tapering growth potential, a select population continued to proliferate, making a major contribution in sustaining the biliary expansion. Our study has highlighted a unique mode of epithelial tissue dynamics, which depends not on a hierarchical system driven by fixated stem cells, but rather, on a stochastically maintained progenitor population with persistent proliferative activity.

Development of a rapid and reversible photocrosslinking of RNA.

We describe a novel reversible interstrand photocrosslinking reaction of RNA. In this system, a modified oligodeoxynucleotide (ODN) containing 3-cyanovinylcarbazole nucleoside ((CNV)K) reacts by photoirradiation at 366 nm for 1 s with pyrimidine residue of a complementary template RNA to yield a crosslinked product in 94% yield.

A selective and sensitive detection of SNP between rice cultivars by using DNA photoligation.

We describe a selective and sensitive single-nucleotide polymorphism (SNP) typing of genes from rice cultivars by using DNA photoligation. This SNP typing method facilitates qualitative determination of genes from indica and japonica rice, and showed a high degree of single nucleotide specificity.

High-contrast, high-intensity laser pulse generation using a nonlinear preamplifier in a Ti:sapphire laser system.

We report a high-contrast, high-intensity Ti:sapphire chirped-pulse amplification system that incorporates a nonlinear preamplifier based on optical parametric chirped-pulse amplification (OPCPA). By cooling the Ti:sapphire crystal in the final amplifier down to 77 K, the chirped-pulses are amplified to 2.9 J at a 10 Hz repetition rate without a thermal lensing effect. Pulse compression down to 19 fs duration obtained after amplification indicates a peak power of 80 TW. With the OPCPA, the temporal contrast is significantly improved to better than 7x10(-9) in a few picoseconds interval prior to the main laser pulse.