Chromatin Modifier EP400 Regulates Oocyte Quality and Zygotic Genome Activation in Mice.

Epigenetic modifiers that accumulate in oocytes, play a crucial role in steering the developmental program of cleavage embryos and initiating life. However, the identification of key maternal epigenetic regulators remains elusive. In the findings, the essential role of maternal Ep400, a chaperone for H3.3, in oocyte quality and early embryo development in mice is highlighted. Depletion of Ep400 in oocytes resulted in a decline in oocyte quality and abnormalities in fertilization. Preimplantation embryos lacking maternal Ep400 exhibited reduced major zygotic genome activation (ZGA) and experienced developmental arrest at the 2-to-4-cell stage. The study shows that EP400 forms protein complex with NFYA, occupies promoters of major ZGA genes, modulates H3.3 distribution between euchromatin and heterochromatin, promotes transcription elongation, activates the expression of genes regulating mitochondrial functions, and facilitates the expression of rate-limiting enzymes of the TCA cycle. This intricate process driven by Ep400 ensures the proper execution of the developmental program, emphasizing its critical role in maternal-to-embryonic transition.

Observing cosmic-ray extensive air showers with a silicon imaging detector.

Extensive air showers induced from high-energy cosmic rays provide a window into understanding the most energetic phenomena in the universe. We present a new method for observing these showers using the silicon imaging detector Subaru Hyper Suprime-Cam (HSC). This method has the advantage of being able to measure individual secondary particles. When paired with a surface detector array, silicon imaging detectors like Subaru HSC will be useful for studying the properties of extensive air showers in detail. The following report outlines the first results of observing extensive air showers with Subaru HSC. The potential for reconstructing the incident direction of primary cosmic rays is demonstrated and possible interdisciplinary applications are discussed.

A simple, rapid, and efficient method for generating multigene-knockout culture cells by the CRISPR/Cas9 system.

We evaluated the efficacy of simultaneous multiple-gene knockout in human culture cells. By simple co-transfection of HeLa cells with a mixture of pX330-based targeting plasmids together with a puromycin resistance plasmid, followed by transient selection of puromycin-resistant cells, Cas9/single-guide RNA (sgRNA)-transduced polyclonal cell populations were selected and grown. Western blot analyses revealed co-transfection of up to seven targeting plasmids for p38α, p38ß, JNK1, JNK2, Mnk1, ERK1, and mLST8 genes, drastically reduced protein expression of these genes in the polyclonal population. Analyses of a randomly isolated group of 25 clones revealed knockout efficiencies for the seven targeted genes ranging between 68% and 100%, and in six clones (24%), all targeted genes were disrupted. Deep sequencing analyses of the individual target sites revealed that, in most cases, Cas9/sgRNA-induced nonhomologous end joining resulted in deletion or insertion of only a few base pairs at the break points. These results demonstrate that simple co-transfection-based simultaneous targeting offers an easy, rapid, and efficient method to generate multiplex gene-knockout cell lines.

Redundant roles of extra-cellular signal-regulated kinase (ERK) 1 and 2 in the G1-S transition and etoposide-induced G2/M checkpoint in HCT116 cells.

The extracellular signal-regulated kinase (ERK) 1 and 2 intracellular signaling pathways play key roles in a variety of cellular processes, such as proliferation and differentiation. Dysregulation of ERK1/2 signaling has been implicated in many diseases, including cancer. Although ERK1/2 signaling pathways have been extensively studied, controversy remains as to whether ERK1 and ERK2 have specific or redundant functions. In this study, we examined the functional roles of ERK1 and ERK2 in cell proliferation and cell cycle progression using an auxin-inducible degron system combined with gene knockout technology. We found that ERK1/2 double depletion, but not ERK1 or ERK2 depletion, substantially inhibited the proliferation of HCT116 cells during G1-S transition. We further demonstrated that ERK1/2-double-depleted cells were much more tolerant to etoposide-induced G2/M arrest than ERK1 or ERK2 single-knockout cells. Together, these results strongly suggest the functional redundancy of ERK1 and ERK2 in both the G1-S transition under physiological conditions and the DNA damage-induced G2/M checkpoint. Our findings substantially advance understanding of the ERK1/2 pathways, which could have strong implications for future pharmacological developments.

Functional Expression of the P2X7 ATP Receptor Requires Eros.

In response to extracellular ATP, the purinergic receptor P2X7 mediates various biological processes, including phosphatidylserine (PtdSer) exposure, phospholipid scrambling, dye uptake, ion transport, and IL-1ß production. A genome-wide CRISPR screen for molecules responsible for ATP-induced PtdSer exposure identified a transmembrane protein, essential for reactive oxygen species (Eros), as a necessary component for P2X7 expression. An Eros-null mouse T cell line lost the ability to expose PtdSer, to scramble phospholipids, and to internalize a dye YO-PRO-1 and Ca2+ ions. Eros-null mutation abolished the ability of an LPS-primed human THP-1 macrophage cell line and mouse bone marrow-derived macrophages to secrete IL-1ß in response to ATP. Eros is localized to the endoplasmic reticulum and functions as a chaperone for NADPH oxidase components. Similarly, Eros at the endoplasmic reticulum transiently associated with P2X7 to promote the formation of a stable homotrimeric complex of P2X7. These results indicated that Eros acts as a chaperone not only for NADPH oxidase, but also for P2X7, and contributes to the innate immune reaction.

Ep400 deficiency in Schwann cells causes persistent expression of early developmental regulators and peripheral neuropathy.

Schwann cells ensure efficient nerve impulse conduction in the peripheral nervous system. Their development is accompanied by defined chromatin changes, including variant histone deposition and redistribution. To study the importance of variant histones for Schwann cell development, we altered their genomic distribution by conditionally deleting Ep400, the central subunit of the Tip60/Ep400 complex. Ep400 absence causes peripheral neuropathy in mice, characterized by terminal differentiation defects in myelinating and non-myelinating Schwann cells and immune cell activation. Variant histone H2A.Z is differently distributed throughout the genome and remains at promoters of Tfap2a, Pax3 and other transcriptional regulator genes with transient function at earlier developmental stages. Tfap2a deletion in Ep400-deficient Schwann cells causes a partial rescue arguing that continued expression of early regulators mediates the phenotypic defects. Our results show that proper genomic distribution of variant histones is essential for Schwann cell differentiation, and assign importance to Ep400-containing chromatin remodelers in the process.

Chromatin remodeler Ep400 ensures oligodendrocyte survival and is required for myelination in the vertebrate central nervous system.

Differentiating oligodendrocytes generate myelin to ensure rapid saltatory conduction in the vertebrate central nervous system. Although oligodendroglial differentiation and myelination are accompanied by dramatic chromatin reorganizations, previously studied chromatin remodelers had only limited direct effects on the process. To study the functional significance of chromatin changes for myelination and identify relevant remodelers, we deleted Ep400, the central ATP-hydrolyzing subunit of the TIP60/EP400 complex, at defined times of mouse oligodendrocyte development. Whereas Ep400-deficient oligodendrocyte precursors develop normally, terminal differentiation and myelination are dramatically impaired. Mechanistically, Ep400 interacts with transcription factor Sox10, binds to regulatory regions of the Myrf gene and is required to induce this central transcriptional regulator of the myelination program. In addition to reduced and aberrant myelin formation, oligodendrocytes exhibit increased DNA damage and apoptosis so that numbers never reach wildtype levels during the short lifespan of Ep400-deficient mice. Ep400 deletion in already mature oligodendrocytes remains phenotypically inapparent arguing that Ep400 is dispensable for myelin maintenance. Given its essential function in myelin formation, modulation of Ep400 activity may be beneficial in conditions such as multiple sclerosis where this process is compromised.

TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets.

Phosphatidylserine (PtdSer) exposure on the surface of activated platelets requires the action of a phospholipid scramblase(s), and serves as a scaffold for the assembly of the tenase and prothrombinase complexes involved in blood coagulation. Here, we found that the activation of mouse platelets with thrombin/collagen or Ca(2+) ionophore at 20 °C induces PtdSer exposure without compromising plasma membrane integrity. Among five transmembrane protein 16 (TMEM16) members that support Ca(2+)-dependent phospholipid scrambling, TMEM16F was the only one that showed high expression in mouse platelets. Platelets from platelet-specific TMEM16F-deficient mice exhibited defects in activation-induced PtdSer exposure and microparticle shedding, although α-granule and dense granule release remained intact. The rate of tissue factor-induced thrombin generation by TMEM16F-deficient platelets was severely reduced, whereas thrombin-induced clot retraction was unaffected. The imaging of laser-induced thrombus formation in whole animals showed that PtdSer exposure on aggregated platelets was TMEM16F-dependent in vivo. The phenotypes of the platelet-specific TMEM16F-null mice resemble those of patients with Scott syndrome, a mild bleeding disorder, indicating that these mice may provide a useful model for human Scott syndrome.

Removal of Pb(II) from water using keratin colloidal solution obtained from wool.

The aim of this study is to investigate the use of keratin colloidal solution, which was obtained from wool, for the removal of Pb(II) from water. The addition of keratin colloidal solution (15 g L(-1), 0.30 mL) to a Pb(II) solution (1.0 mM, 0.90 mL, pH 5.0) resulted in the formation and precipitation of a Pb-keratin aggregate. Measurement of the Pb(II) and protein concentrations in the supernatant solution revealed that 88 and 99 % of the Pb(II) and keratin protein were removed from the solution, respectively. The maximum Pb(II) uptake capacity of keratin in the colloidal solution was 43.3 mg g(-1). In addition, the Pb-keratin aggregate was easily decomposed via the addition of nitric acid, which enabled the recovery of Pb(II). However, aggregation did not occur in solutions with Pb(II) concentrations below 0.10 mM. Therefore, we used a keratin colloidal solution encapsulated in a dialysis cellulose tube to remove Pb(II) from 0.10 mM solutions, which enabled the removal of 95 % of the Pb(II). From these results, we conclude that keratin colloidal solution is useful for the treatment of water polluted with Pb(II).

Calcium-dependent phospholipid scramblase activity of TMEM16 protein family members.

BACKGROUND: TMEM16A and 16B work as Cl(-) channel, whereas 16F works as phospholipid scramblase. The function of other TMEM16 members is unknown. RESULTS: Using TMEM16F(-/-) cells, TMEM16C, 16D, 16F, 16G, and 16J were shown to be lipid scramblases. CONCLUSION: Some TMEM16 members are divided into two Cl(-) channels and five lipid scramblases. SIGNIFICANCE: Learning the biochemical function ofTMEM16family members is essential to understand their physiological role. Asymmetrical distribution of phospholipids between the inner and outer plasma membrane leaflets is disrupted in various biological processes. We recently identified TMEM16F, an eight-transmembrane protein, as a Ca(2+)-dependent phospholipid scramblase that exposes phosphatidylserine (PS) to the cell surface. In this study, we established a mouse lymphocyte cell line with a floxed allele in the TMEM16F gene. When TMEM16F was deleted, these cells failed to expose PS in response to Ca(2+) ionophore, but PS exposure was elicited by Fas ligand treatment. We expressed other TMEM16 proteins in the TMEM16F(-/-) cells and found that not only TMEM16F, but also 16C, 16D, 16G, and 16J work as lipid scramblases with different preference to lipid substrates. On the other hand, a patch clamp analysis in 293T cells indicated that TMEM16A and 16B, but not other family members, acted as Ca(2+)-dependent Cl(-) channels. These results indicated that among 10 TMEM16 family members, 7 members could be divided into two subfamilies, Ca(2+)-dependent Cl(-) channels (16A and 16B) and Ca(2+)-dependent lipid scramblases (16C, 16D, 16F, 16G, and 16J).

Early rehabilitation with weight-bearing standing-shaking-board exercise in combination with electrical muscle stimulation after anterior cruciate ligament reconstruction.

The objective of early rehabilitation after anterior cruciate ligament (ACL) reconstruction is to increase the muscle strength of the lower extremities. Closed kinetic chain (CKC) exercise induces co-contraction of the agonist and antagonist muscles. The purpose of this study was to compare the postoperative muscle strength/mass of subjects who performed our new CKC exercise (new rehabilitation group:group N) from week 4, and subjects who received traditional rehabilitation alone (traditional rehabilitation group:group T). The subjects stood on the device and maintained balance. Then, low-frequency stimulation waves were applied to 2 points each in the anterior and posterior region of the injured thigh 3 times a week for 3 months. Measurement of muscle strength was performed 4 times (before the start, and then once a month). Muscle mass was evaluated in CT images of the extensor and flexor muscles of 10 knees (10 subjects) in each group. The injured legs of group N showed significant improvement after one month compared to group T. The cross-sectional area of the extensor muscles of the injured legs tended to a show a greater increase at 3 months in group N. This rehabilitation method makes it possible to contract fast-twitch muscles, which may be a useful for improving extensor muscle strength after ACL reconstruction.

Thermal analysis of polyethylene glycol: evolved gas analysis with ion attachment mass spectrometry.

The thermal decomposition of polyethylene glycol was investigated by using a technique combining evolved gas analysis (time-resolved pyrolysis) with ion-attachment mass spectrometry. This technique allows the detection of intact pyrolysis products and, therefore, offers the opportunity for direct real-time monitoring of thermal by-products. Unstable products can thus be detected; for instance, many highly reactive organic peroxides, such as CH(3)OOH and HOCH(2)OOH, were found in this study. Classification analysis revealed 10 major compositional formulas among the product species: C(n)H(2)(n)(+2)O, C(n)H(2)(n)(+2)O(2), C(n)H(2)(n)(+2)O(3), C(n)H(2)(n)(+2)O(4), C(n)H(2)(n)(+2)O(5), C(n)H(2)(n)(+2)O(6), C(n)H(2)(n)(+2)O(7), C(n)H(2)(n)O, C(n)H(2)(n)O(2), and HO(CH(2)CH(2)O)(n)H ethylene glycol oligomers. The Li(+) ion adduct mass spectra showed a characteristic profile in terms of both the appearance of unique components and the distribution of pyrolysis products. Among the products of the thermal decomposition of PEG, formaldehyde (HCHO) and organic peroxides were particularly interesting. Formaldehyde, one of the 10 most abundant products, is a known human carcinogen. The detection of peroxides suggests that they may form during the incineration of PEG, which may have important environmental implications. The existence of peroxide products may have implications for chemical evolution in incinerator systems.

Thermal decomposition of pyridoxine: an evolved gas analysis-ion attachment mass spectrometry study.

RATIONALE: Pyridoxine is an important vitamer in food and pharmaceutical products. Heat treatments applied during preparation or storage of the products cause the decomposition of pyridoxine. Identification and understanding of the degradation products of pyridoxine and studying its decomposition kinetics are essential in the preparation and preservation of pyridoxine-containing foods and pharmaceuticals. METHODS: Real-time, non-isothermal decomposition of pyridoxine was studied using evolved gas analysis-Li(+) ion attachment mass spectrometry (EGA-Li(+) IAMS). Arrhenius parameters for the thermal decomposition of pyridoxine were obtained via the total ion monitoring (TIM) curve. RESULTS: Most of the pyridoxine evaporated in molecular form, but the formation of pyridoxal and o-quinone methide, both biologically important species, was also observed from the solid-phase degradation of pyridoxine. The observation of o-quinone methide, a species possessing anticancer activity, was particularly noteworthy due to its chemical instability. The activation energy (E(a) ) for pyridoxine decomposition determined by EGA-IAMS was found to be 20.0 kcal mol(-1) , and the pre-exponential factor (A) was 5.7 × 10(9) min(-1) . CONCLUSIONS: The calculated kinetic parameters are important for predicting the thermal stability of pyridoxine vitamer. The estimated lifetime (t(90%,25°C) ) of 1.7 × 10(-2) years in nitrogen was also obtained from the EGA-IAMS experiment.

Design and performance of a compact Li+ ion attachment mass spectrometry system with an atmospheric sampling device.

This report describes the development of a compact ion attachment mass spectrometry system. A single turbomolecular pump was employed to fill the basic requirements for vacuum conditions simply and cost-effectively, without the need for a differential pumping stage. A Li(+) ion source was placed in the first of two vacuum chambers; a 0.4 mm aperture allowed the product ions to enter the second chamber for mass analysis. With the present system, any chemical species, including radical intermediates, can be detected at atmospheric pressure in real-time. The minimum detectable amount (at S/N = 3) of toluene was around 1.3 × 10(-12) g/s with a linearity greater than 10(4). For illustrative purposes, we tested the system on laboratory air and on aroma compounds in the headspace of the Yuzu plant, Citrus junos.

Thermal stability of vitamin C: thermogravimetric analysis and use of total ion monitoring chromatograms.

The thermal decomposition kinetics and shelf life of vitamin C in nitrogen or air were studied by using thermogravimetric analysis (TGA) and evolved-gas analysis-lithium-ion attachment mass spectrometry (EGA-Li⁺IAMS). Arrhenius parameters obtained via TGA were reported for thermal decomposition. For vitamin C in a nitrogen atmosphere, the activation energy (E(a)) was 25.1 kcal/mol and the pre-exponential factor (A) was 2.5 × 10¹¹ min⁻¹. The kinetic parameters estimated via TGA agreed with values estimated from a pyrogram when the weight loss observed by TGA was shown to be due to gas evolution as a result of decomposition of the compound. Thermal stability was expressed by calculating the time for 10% of the vitamin C to decompose at 25 °C (t(90%,25 °C)). The t(90%,25 °C) for vitamin C obtained via TGA or EGA-Li⁺IAMS was higher in nitrogen (2.0 and 2.0 years, respectively) than in air (1.3 and 1.6 years, respectively). This indicates that the type of atmosphere influences vitamin C stability.

Evolved gas analysis of Ti(C5H5)2Cl2 by means of Li+ ion attachment mass spectrometry.

Characterization of the compound Ti(C(5)H(5))(2)Cl(2) was studied using Li(+) ion attachment mass spectrometry (IAMS) as an analytical methodology. Since this target compound is used as an anticancer drug in the treatment of leukemia, accurate and rapid monitoring methods for the determination of titanium drugs in a hospital environment are desirable. A quadrupole mass spectrometry system along with a Li(+) ion attachment technique and a direct inlet probe (DIP) produced the Li(+) adduct of Ti(C(5)H(5))(2)Cl(2), Ti(C(5)H(5))(2)Cl(2)Li(+). The DIP also was used to study the temperature-resolved behavior of this compound. The slope of the plot of signal intensity of Ti(C(5)H(5))(2)Cl(2)Li(+) versus temperature for Ti(C(5)H(5))(2)Cl(2) sublimation from 60 to 100 °C was used to determine an apparent activation energy (E(a)) of 124.43 kJ/mol for the sublimation of Ti(C(5)H(5))(2)Cl(2). This value is comparable to the reported value of 118.8 kJ/mol for molar enthalpy of sublimation of Ti(C(5)H(5))(2)Cl(2). These results demonstrate that the IAMS methodology can be used to study the enthalpy of sublimation for d-metal complex materials.

Is the bisphenol A biradical formed in the pyrolysis of polycarbonate?

An unknown species has been detected in the analysis of the products in a pyrolysis of polycarbonate using Li(+) ion-attachment mass spectrometry (IAMS). The mass spectra exhibited a Li(+) adduct peak at m/z 233 that was tentatively assigned to bisphenol A (BPA) biradical. Experimentally, this assignment was supported by the observation that the production rate increased under an inert nitrogen atmosphere. To further confirm the assignment, the stability of the BPA biradical to intramolecular rearrangement reactions as well as unimolecular decomposition has been analyzed via density functional theory calculations [B3LYP/6-311+G(3df,2p)]. The results show that the bisphenol A biradical is an open-shell biradical singlet that is stable to unimolecular decomposition. Although some of the proposed intramolecular rearrangement products have lower energies than those of the BPA diradical, these pathways have large reaction barriers and the kinetic lifetime of the radical is expected to be of the order of hours under the conditions of the experiment. The calculations also reveal that the bisphenol A diradical has large Li(+) affinities supporting the fact that these Li(+) complexes could be detected in the Li(+) ion attachment mass spectrometry. On the basis of these results the Li(+) adduct peak at m/z 233 detected in the pyrolysis of polycarbonate is assigned to the bisphenol A biradical.

Thermal decomposition of vitamin C: An evolved gas analysis-ion attachment mass spectrometry study.

Evolved gas analysis-ion attachment mass spectrometry (EGA-IAMS) was utilised to study the real-time non-isothermal decomposition of vitamin C. Dehydro-l-ascorbic acid, which has until this study been undetectable from the solid phase degradation of vitamin C, was observed as a decomposition product. While it is an important compound because it possesses some biological activity, dehydro-l-ascorbic acid is difficult to measure due to its chemical instability. In the present study using EGA-IAMS, we were able to detect dehydro-l-ascorbic acid from the thermal degradation of vitamin C. Our EGA-IAMS results obtained from the thermal decomposition of vitamin C were compared with a previous study employing pyrolysis-gas chromatography-mass spectrometry (Pyr-GC-MS). The observed quantitative and qualitative differences of the pyrolysis products obtained by the two techniques (EGA-IAMS vs. Pyr-GC-MS) are in part due to the difference in transportation time of the products out of the pyrolysis chamber.

Design and performance of an evolved gas analysis ion attachment mass spectrometer.

We designed a simple evolved gas analysis (EGA) system to act as a sampler between solid samples at atmospheric pressure and the high vacuum inside a mass spectrometer. The newly designed stainless steel system is simple, small and rugged and fulfills all the basic requirements for EGA. The temperature is programmable with 60 degrees C/min as the maximum heating rate and the temperature range is up to 600 degrees C. With this system coupled with lithium ion attachment mass spectrometry (IAMS), it is possible to study the temperature-programmed decomposition of a number of solid materials by detecting any chemical species on a real-time basis. For illustrative purposes, EGA-IAMS experiments of polyethylene polymers have been conducted.

Formation of bisphenol A by thermal degradation of poly(bisphenol A carbonate).

The thermal decomposition of poly(bisphenol A carbonate) (PoC) results in the formation of the endocrine disruptor bisphenol A (BPA). In the present work, we investigated the kinetics of the thermal decomposition of PoC, and the subsequent decomposition of BPA, under pyrolysis conditions and in the presence of oxygen by using infrared image furnace-ion attachment mass spectrometry. The decomposition of PoC obeyed Arrhenius kinetics, which allowed us to determine the activation energy (E(a)) for thermal decomposition to BPA from Arrhenius plots. From the selected ion monitoring curves for BPA, E(a) for thermal decomposition in a nitrogen atmosphere was calculated to be 133.2 kcal mol(-1), whereas E(a) for oxidative thermal decomposition was calculated to be approximately 35% lower (86.5 kcal mol(-1)).