Lack of Cul4b, an E3 Ubiquitin Ligase Component, Leads to Embryonic Lethality and Abnormal Placental Development

Cullin-RING ligases (CRLs) complexes participate in the regulation of diverse cellular processes, including cell cycle progression, transcription, signal transduction and development. Serving as the scaffold protein, cullins are crucial for the assembly of ligase complexes, which recognize and target various substrates for proteosomal degradation. Mutations in human CUL4B, one of the eight members in cullin family, are one of the major causes of X-linked mental retardation. We here report the generation and characterization of Cul4b knockout mice, in which exons 3 to 5 were deleted. In contrast to the survival to adulthood of human hemizygous males with CUL4B null mutation, Cul4b null mouse embryos show severe developmental arrest and usually die before embryonic day 9.5 (E9.5). Accumulation of cyclin E, a CRL (CUL4B) substrate, was observed in Cul4b null embryos. Cul4b heterozygotes were recovered at a reduced ratio and exhibited a severe developmental delay. The placentas in Cul4b heterozygotes were disorganized and were impaired in vascularization, which may contribute to the developmental delay. As in human CUL4B heterozygotes, Cul4b null cells were selected against in Cul4b heterozygotes, leading to various degrees of skewed X-inactivation in different tissues. Together, our results showed that CUL4B is indispensable for embryonic development in the mouse

Theoretical analysis and enhanced nitrogen removal performance of step-feed SBR

To achieve high nitrification and denitrification rates, step-feed SBR with multiple aerobic and anoxic phases was adopted to study nitrogen removal performance. Theoretical analysis of step-feed SBR was presented first, from which feeding steps and C/N ratio had significant influences on nitrogen removal performance. Total nitrogen removal efficiency would increase with increasing of feeding steps, while the increasing extent was not distinct with feeding steps above 4. At a given feeding step, nitrogen removal efficiency would also increase with increasing of C/N ratio. Experimental work was conducted in a lab-scale SBR to investigate practical effects of these critical factors, using real municipal wastewater. The results showed when C/N ratio was appropriately decreasing influent flow mode could achieve enhanced nitrogen removal with less adding of external carbon source, compared with equal influent flow mode. Three-step equal influent flow mode was recommended to treat common municipal wastewater in view of operation complexity. Non steady-state experiments over about three months confirmed step-feed SBR was an enhanced nitrogen removal process with high efficiency. Total nitrogen in the effluent was under 2 mg/L, the average removal efficiency achieved for TN was over 97% just adding a little external carbon source, and good sludge settleability was obtained

Theoretical analysis and enhanced nitrogen removal performance of step-feed SBR

To achieve high nitrification and denitrification rates, step-feed SBR with multiple aerobic and anoxic phases was adopted to study nitrogen removal performance. Theoretical analysis of step-feed SBR was presented first, from which feeding steps and C/N ratio had significant influences on nitrogen removal performance. Total nitrogen removal efficiency would increase with increasing of feeding steps, while the increasing extent was not distinct with feeding steps above 4. At a given feeding step, nitrogen removal efficiency would also increase with increasing of C/N ratio. Experimental work was conducted in a lab-scale SBR to investigate practical effects of these critical factors, using real municipal wastewater. The results showed when C/N ratio was appropriately decreasing influent flow mode could achieve enhanced nitrogen removal with less adding of external carbon source, compared with equal influent flow mode. Three-step equal influent flow mode was recommended to treat common municipal wastewater in view of operation complexity. Non steady-state experiments over about three months confirmed step-feed SBR was an enhanced nitrogen removal process with high efficiency. Total nitrogen in the effluent was under 2 mg/L, the average removal efficiency achieved for TN was over 97% just adding a little external carbon source, and good sludge settleability was obtained

A Test Method for Acoustic Emission Properties of Natural Cellulose Fiber-Reinforced Composites

To test the acoustic performance of fiber-reinforced composites for replacing wood, an acoustic vibration test method is developed. For evaluation of the test method, composites are manufactured using hemp and ramie embedded in epoxy, through vacuum-assisted resin infusion molding. The effects of the most important factors, i.e., impulse, relative humidity (RH), and specimen thickness, on the acoustic vibration response of the composites are systematically studied. The magnitudes of the impulses, represented by different masses of the dropping balls, seem to have little influence on the shapes of the acoustic vibration curves, although the intensity of the spectra increases as the impulse increases. The RH influences the spectrum shape significantly due to variation in the Young’s modulus and density of the material upon absorption of moisture. The specimen thickness also greatly affects the testing results. The specific dynamic modulus, acoustic radiation damping coefficient, and acoustic impedance change a little as the impulse magnitude and RH change, but decrease substantially as the specimen thickness increases. The specific dynamic modulus can be linearly correlated with the flexural modulus of a material

Mutation in CUL4B, Which Encodes a Member of Cullin-RING Ubiquitin Ligase Complex, Causes X-Linked Mental Retardation

We reevaluated a previously reported family with an X-linked mental retardation syndrome and attempted to identify the underlying genetic defect. Screening of candidate genes in a 10-Mb region on Xq25 implicated CUL4B as the causative gene. CUL4B encodes a scaffold protein that organizes a cullin-RING (really interesting new gene) ubiquitin ligase (E3) complex in ubiquitylation. A base substitution, c.1564C→T, converted a codon for arginine into a premature termination codon, p.R388X, and rendered the truncated peptide completely devoid of the C-terminal catalytic domain. The nonsense mutation also results in nonsense-mediated mRNA decay in patients. In peripheral leukocytes of obligate carriers, a strong selection against cells expressing the mutant allele results in an extremely skewed X-chromosome inactivation pattern. Our findings point to the functional significance of CUL4B in cognition and in other aspects of human development

Where to place methane monitoring sites in China to better assist carbon management

Abstract Methane (CH4) is the second most potent greenhouse gas (GHG), and China emerges as the largest anthropogenic CH4 emitter by country. Current limited CH4 monitoring systems in China are unfortunately inadequate to support carbon management. Here we use the Weather Research and Forecasting model (WRF) coupled with a GHG module and satellite constrained emissions to simulate the spatiotemporal distribution of CH4 over East Asia in 2017. Model evaluations using both satellite retrievals and ground-based observations indicate reliable performance. We further inter-compare four proper orthogonal decomposition (POD)-based sensor placement algorithms and find they are able to capture main spatial features of surface CH4 under an oversampled condition. The QR pivot algorithm exhibits superiority in capturing high CH4, and it offers the best reconstruction with both high efficiency and accuracy. Areas with high CH4 concentrations and intense anthropogenic activities remain underrepresented by current CH4 sampling studies, leading to notable reconstruction error over central and eastern China. Optimal planning of 160 sensors guided by the QR pivot algorithm can yield reasonable reconstruction performance and costs of site construction. Our results can provide valuable references for future planning of CH4 monitoring sites

Successive Waste as Reagent: Two More Steps Forward in a Pinnick Oxidation

The successful development of the classical Pinnick oxidation into a new and promising oxidative lactonization reaction is reported. Chiral 3-oxindolepropionic aldehydes, Michael adducts of 3-olefinic oxindoles with aliphatic aldehydes, are directly converted to spirocyclic oxindole-γ-lactones solely by sodium chlorite via a tandem Pinnick oxidation/chlorination/substitution sequence. This reaction uses waste ClO^– generated in the initial Pinnick oxidation as an ecofriendly halogenating agent for the subsequent chlorination, and then it utilizes the byproduct OH^– formed in the chlorination to facilitate the final internal substitution

Characterization of X chromosome inactivation by Cul4b expression in heterozygous mice.

<p>(A–C) Percentages of cells positive for Cul4b of <i>Cul4b</i> heterozygous mice and littermate wild-type female controls at 4 months (A), 3 weeks (B) and newborn (C). More than 2,000 cells of each tissue were scored. Hi, hippocampus; Ki, kidney; Li, liver; Lu, lung. Data were presented as mean±SD. *: p<0.05; **: p<0.01; ***: p<0.001. (D–E) Representative images of liver (D) and hippocampus (E) at 3 weeks stained with an antibody against Cul4b. Sections were counterstained with haematoxylin. Lower panels are the higher magnification of the upper panels. (F) Immunohistochemistry of paraffin sections of <i>Cul4b</i> heterozygous embryos at 7.5 dpc with an anti-Cul4b antibody. Embryos at 7.5 dpc were paraffin embedded and cross sectioned together with their surrounding deciduas. Middle and right panels are the higher magnification of the left panel.</p

Generation of <i>Cul4b</i> flox mice.

<p>(A) Strategy for generation of <i>Cul4b</i> floxed targeting vector. On the top was shown the wild-type allele of <i>Cul4b</i> gene. The targeting vector and targeted allele were shown in the middle and at the bottom, respectively. <i>BamHI</i> (labeled B) and <i>XbaI</i> (labeled X) sites are indicated. Black bars indicating the positions of the probes used in Southern blots are also indicated. (B) Southern blot analysis of genomic DNA isolated from wild-type ES cells and selected ES cell clones. <i>BamHI</i> digested DNA was hybridized with 5′ probe and <i>XbaI</i> digested DNA was hybridized with 3′ probe. WT, wild-type allele; F, flox allele. (C) cDNA sequencing of <i>Cul4b</i> gene from the brain tissue of brain-specific knockout mice (<i>Cul4b</i>^flox/Y;<i>Nesin-Cre</i>). As predicted, exon 2 was spliced onto exon 6 after the excision of exons 3–5. (D) Real-time RT-PCR analysis of <i>Cul4b</i> mRNA isolated from brain tissues of wild-type males (<i>Cul4b</i>^+/Y;<i>Nestin-Cre</i>^+/−), wild-type females (<i>Cul4b^+/+</i>;<i>Nestin-Cre</i>^+/−), heterozygous females (<i>Cul4b</i>^+/flox;<i>Nestin-Cre</i>^+/−), and conditional knock-out males (<i>Cul4b</i>^flox/Y;<i>Nestin-Cre</i>^+/−). (E) Western blot analysis of Cul4b protein isolated from brain tissues of wild-type, heterozygous and conditional knockout mice using an anti-Cul4b antibody. Gapdh was used as a loading control.</p