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  • 1
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    CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation (2015)
    American Association for the Advancement of Science (AAAS)
    Details
    Publication Date: 2015-08-16
    Description: The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9–mediated knockin system.
    Electronic ISSN: 2375-2548
    Topics: Natural Sciences in General
    Published by American Association for the Advancement of Science (AAAS)
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  • 2
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    Selectively Constrained RNA Editing Regulation Crosstalks with piRNA Biogenesis in Primates (2015)
    Oxford University Press
    Details
    Publication Date: 2015-11-20
    Description: Although millions of RNA editing events have been reported to modify hereditary information across the primate transcriptome, evidence for their functional significance remains largely elusive, particularly for the vast majority of editing sites in noncoding regions. Here, we report a new mechanism for the functionality of RNA editing—a crosstalk with PIWI-interacting RNA (piRNA) biogenesis. Exploiting rhesus macaque as an emerging model organism closely related to human, in combination with extensive genome and transcriptome sequencing in seven tissues of the same animal, we deciphered accurate RNA editome across both long transcripts and the piRNA species. Superimposing and comparing these two distinct RNA editome profiles revealed 4,170 editing-bearing piRNA variants, or epiRNAs, that primarily derived from edited long transcripts. These epiRNAs represent distinct entities that evidence an intersection between RNA editing regulations and piRNA biogenesis. Population genetics analyses in a macaque population of 31 independent animals further demonstrated that the epiRNA-associated RNA editing is maintained by purifying selection, lending support to the functional significance of this crosstalk in rhesus macaque. Correspondingly, these findings are consistent in human, supporting the conservation of this mechanism during the primate evolution. Overall, our study reports the earliest lines of evidence for a crosstalk between selectively constrained RNA editing regulation and piRNA biogenesis, and further illustrates that such an interaction may contribute substantially to the diversification of the piRNA repertoire in primates.
    Print ISSN: 0737-4038
    Electronic ISSN: 1537-1719
    Topics: Biology
    Published by Oxford University Press
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  • 3
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    Global existence of solution of Cauchy problem for a non-linear wave equation (2013)
    Oxford University Press
    Details
    Publication Date: 2013-09-27
    Description: In this paper, we prove that the Cauchy problem for a non-linear wave equation (1) has a unique global generalized solutions in and a unique global classical solution in . We also prove that the Cauchy problem for the equation (1) admits a unique global generalized solution in C 2 ([0, ); W m , p ( R )   L ( R ))( m  ≥ 0 is an integer,1 ≤  p  ≤ ) and a unique global classical solution in .
    Print ISSN: 0272-4960
    Electronic ISSN: 1464-3634
    Topics: Mathematics
    Published by Oxford University Press
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  • 4
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    Structural insight into the mechanism of stabilization of the 7SK small nuclear RNA by LARP7 (2015)
    Oxford University Press
    Details
    Publication Date: 2015-04-02
    Description: The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3'-end of 7SK. The structure shows that the penultimate uridine is tethered by the two domains, the La-motif and the RNA-recognition motif (RRM1), and reveals that the RRM1 is significantly smaller and more exposed than in the La protein. Sequence analysis suggests that this impacts interaction with 7SK. Binding assays, footprinting and small-angle scattering experiments show that a second RRM domain located at the C-terminus binds the apical loop of the 3' hairpin of 7SK, while the N-terminal domains bind at its foot. Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5'-end with the foot of the 3' hairpin and has thus functional implications.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 5
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    Teosinte ligule allele narrows plant architecture and enhances high-density maize yields (2019)
    American Association for the Advancement of Science (AAAS)
    In: Science
    Details
    Publication Date: 2019
    Description: 〈p〉Increased planting densities have boosted maize yields. Upright plant architecture facilitates dense planting. Here, we cloned 〈i〉UPA1〈/i〉 (〈i〉Upright Plant Architecture1〈/i〉) and 〈i〉UPA2〈/i〉, two quantitative trait loci conferring upright plant architecture. 〈i〉UPA2〈/i〉 is controlled by a two-base sequence polymorphism regulating the expression of a B3-domain transcription factor (〈i〉ZmRAVL1〈/i〉) located 9.5 kilobases downstream. 〈i〉UPA2〈/i〉 exhibits differential binding by DRL1 (DROOPING LEAF1), and DRL1 physically interacts with LG1 (LIGULELESS1) and represses LG1 activation of 〈i〉ZmRAVL1〈/i〉. 〈i〉ZmRAVL1〈/i〉 regulates 〈i〉brd1〈/i〉 (〈i〉brassinosteroid C-6 oxidase1〈/i〉), which underlies 〈i〉UPA1〈/i〉, altering endogenous brassinosteroid content and leaf angle. The 〈i〉UPA2〈/i〉 allele that reduces leaf angle originated from teosinte, the wild ancestor of maize, and has been lost during maize domestication. Introgressing the wild 〈i〉UPA2〈/i〉 allele into modern hybrids and editing 〈i〉ZmRAVL1〈/i〉 enhance high-density maize yields.〈/p〉
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
    Published by American Association for the Advancement of Science (AAAS)
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  • 6
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    Divergence in Enzymatic Activities in the Soybean GST Supergene Family Provides New Insight into the Evolutionary Dynamics of Whole-Genome Duplicates (2015)
    Oxford University Press
    Details
    Publication Date: 2015-10-18
    Description: Whole-genome duplication (WGD), or polyploidy, is a major force in plant genome evolution. A duplicate of all genes is present in the genome immediately following a WGD event. However, the evolutionary mechanisms responsible for the loss of, or retention and subsequent functional divergence of polyploidy-derived duplicates remain largely unknown. In this study we reconstructed the evolutionary history of the glutathione S-transferase (GST) gene family from the soybean genome, and identified 72 GST duplicated gene pairs formed by a recent Glycine -specific WGD event occurring approximately 13 Ma. We found that 72% of duplicated GST gene pairs experienced gene losses or pseudogenization, whereas 28% of GST gene pairs have been retained in the soybean genome. The GST pseudogenes were under relaxed selective constraints, whereas functional GSTs were subject to strong purifying selection. Plant GST genes play important roles in stress tolerance and detoxification metabolism. By examining the gene expression responses to abiotic stresses and enzymatic properties of the ancestral and current proteins, we found that polyploidy-derived GST duplicates show the divergence in enzymatic activities. Through site-directed mutagenesis of ancestral proteins, this study revealed that nonsynonymous substitutions of key amino acid sites play an important role in the divergence of enzymatic functions of polyploidy-derived GST duplicates. These findings provide new insights into the evolutionary and functional dynamics of polyploidy-derived duplicate genes.
    Print ISSN: 0737-4038
    Electronic ISSN: 1537-1719
    Topics: Biology
    Published by Oxford University Press
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  • 7
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    Biophysical regulations of NEE light response in a steppe and a cropland in Inner Mongolia (2012)
    Oxford University Press
    Details
    Publication Date: 2012-04-13
    Description: Aims Ecosystem carbon models often require accurate net ecosystem exchange of CO 2 (NEE) light-response parameters, which can be derived from the Michaelis–Menten equation. These parameters include maximum net ecosystem exchange (NEE max ), apparent quantum use efficiency ( α ) and daytime ecosystem respiration rate ( R e ). However, little is known about the effects of land conversion between steppe and cropland on these parameters, especially in semi-arid regions. To understand how these parameters vary in responses to biotic and abiotic factors under land conversions, seasonal variation of light-response parameters were evaluated for a steppe and a cropland of Inner Mongolia, China, during three consecutive years (2006–08) with different precipitation amounts. Methods NEE was measured over a steppe and a cropland in Duolun, Inner Mongolia, China, using the eddy covariance technique, and NEE light-response parameters (NEE max , α and R e ) were derived using the Michaelis–Menten model. Biophysical regulations of these parameters were evaluated using a stepwise regression analysis. Important Findings The maximum absolute values of NEE max occurred in the meteorological regimes of 15°C ≤ T a 〈 25°C, vapor pressure deficit (VPD) 〈 1 KPa and 0.21 m 3 m – 3 ≤ volumetric soil water content at 10 cm (SWC) 〈 0.28 m 3 m – 3 for both the steppe and the cropland ecosystems. The variations of α and R e showed no regular variation pattern in different T air , VPD and SWC regimes. Under the same regime of T air , VPD and SWC, the cropland had higher absolute values of NEE max than the steppe. Canopy conductance and leaf area index (LAI) were dominant drivers for variations in NEE light-response parameters of the steppe and the cropland. The seasonal variation of NEE light-response parameters followed the variation of LAI for two ecosystems. The peak values of all light-response parameters for the steppe and the cropland occurred from July to August. The values of NEE light-response parameters (NEE max , α and R e ) were lower in the driest year (2007). Seasonally averaged NEE light-response parameters for the cropland surpassed those for the steppe. Land conversion from steppe to cropland enhanced NEE light-response parameters during the plant growing period. These results will have significant implications for improving the models on regional NEE variation under climate change and land-use change scenarios.
    Print ISSN: 1752-993X
    Electronic ISSN: 1752-9921
    Topics: Biology
    Published by Oxford University Press
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  • 8
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    R-matrix calculations of the photoionization cross-sections of Ti2+ (2015)
    Oxford University Press
    Details
    Publication Date: 2015-09-24
    Description: The photoionization cross-sections of Ti 2+ in the ground state ([Ne]3s 2 3p 6 3d 2 3 F e ) are calculated using both non-relativistic ( LS -coupling) and relativistic (Breit–Pauli) R -matrix methods for the photon energy from 27.49 eV (I.P.) to 48.00 eV. The results show that, in this energy range, the photoionization cross-sections are dominated by resonances 3p 5 3d 3 , 3p 5 3d 2 4d and 3p 5 3d 2 4s, which are labelled by LS -terms by combining with the multiconfiguration Dirac-Fock calculations. Moreover, from the fractional parentage coefficients and the percentages (the square of the configuration interaction expansion coefficients) of the LS -terms involved in the final resonance states, the relative strength of the resonances have been explained qualitatively in a general way. The present results should be of great help in the modelling and diagnostic of astrophysical plasmas as well as laboratory plasmas.
    Print ISSN: 0035-8711
    Electronic ISSN: 1365-2966
    Topics: Physics
    Published by Oxford University Press
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  • 9
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    Temporal changes of antibiotic-resistance genes and bacterial communities in two contrasting soils treated with cattle manure (2016)
    Oxford University Press
    Details
    Publication Date: 2016-01-30
    Description: The emerging environmental spread of antibiotic-resistance genes (ARGs) and their subsequent acquisition by clinically relevant microorganisms is a major threat to public health. Animal manure has been recognized as an important reservoir of ARGs; however, the dissemination of manure-derived ARGs and the impacts of manure application on the soil resistome remain obscure. Here, we conducted a microcosm study to assess the temporal succession of total bacteria and a broad spectrum of ARGs in two contrasting soils following manure application from cattle that had not been treated with antibiotics. High-capacity quantitative PCR detected 52 unique ARGs across all the samples, with β-lactamase as the most dominant ARG type. Several genes of soil indigenous bacteria conferring resistance to β-lactam, which could not be detected in manure, were found to be highly enriched in manure-treated soils, and the level of enrichment was maintained over the entire course of 140 days. The enriched β-lactam resistance genes had significantly positive relationships with the relative abundance of the integrase intI1 gene, suggesting an increasing mobility potential in manure-treated soils. The changes in ARG patterns were accompanied by a significant effect of cattle manure on the total bacterial community compositions. Our study indicates that even in the absence of selective pressure imposed by agricultural use of antibiotics, manure application could still strongly impact the abundance, diversity and mobility potential of a broad spectrum of soil ARGs. Our findings are important for reliable prediction of ARG behaviors in soil environment and development of appropriate strategies to minimize their dissemination.
    Print ISSN: 0168-6496
    Electronic ISSN: 1574-6941
    Topics: Biology
    Published by Oxford University Press
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  • 10
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    Cold-inducible RNA-binding protein CIRP/hnRNP A18 regulates telomerase activity in a temperature-dependent manner (2016)
    Oxford University Press
    Details
    Publication Date: 2016-01-30
    Description: The telomerase is responsible for adding telomeric repeats to chromosomal ends and consists of the reverse transcriptase TERT and the RNA subunit TERC. The expression and activity of the telomerase are tightly regulated, and aberrant activation of the telomerase has been observed in 〉85% of human cancers. To better understand telomerase regulation, we performed immunoprecipitations coupled with mass spectrometry (IP-MS) and identified cold inducible RNA-binding protein (CIRP or hnRNP A18) as a telomerase-interacting factor. We have found that CIRP is necessary to maintain telomerase activities at both 32°C and 37°C. Furthermore, inhibition of CIRP by CRISPR-Cas9 or siRNA knockdown led to reduced telomerase activities and shortened telomere length, suggesting an important role of CIRP in telomere maintenance. We also provide evidence here that CIRP associates with the active telomerase complex through direct binding of TERC and regulates Cajal body localization of the telomerase. In addition, CIRP regulates the level of TERT mRNAs. At the lower temperature, TERT mRNA is upregulated in a CIRP-dependent manner to compensate for reduced telomerase activities. Taken together, these findings highlight the dual roles that CIRP plays in regulating TERT and TERC, and reveal a new class of telomerase modulators in response to hypothermia conditions.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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