Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2

CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly

Identification of transcription-factor genes expressed in the Arabidopsis female gametophyte

Dongfang Wang, Changqing Zhang, David J. Hearn, Il-HO Kang, megan I. Skaggs, Karen S. Schumaker, and Ramin Yadegari are with the School of Plant Sciences, University of Arizona, Tucson, Arizona 85721-0036, USA -- Il-Ho Kang, Jayson A. Punwani, and Gary N. Drews are with the Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840, USA -- Changqing Zhang is with The Section of Molecular, Cell and Developmental Biology, University of Texas at Austin, Austin, Texas 78712-0159, USA -- David J. Hearn is with the Department of Biological Sciences, Towson University, Towson, Maryland 21252-0001, USA -- Il-Ho Kang is with the Department of Horticulture, Iowa State University, Ames, Iowa 50011-1100, USA --Jayson A. Punwani is with the Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280, USABackground In flowering plants, the female gametophyte is typically a seven-celled structure with four cell types: the egg cell, the central cell, the synergid cells, and the antipodal cells. These cells perform essential functions required for double fertilization and early seed development. Differentiation of these distinct cell types likely involves coordinated changes in gene expression regulated by transcription factors. Therefore, understanding female gametophyte cell differentiation and function will require dissection of the gene regulatory networks operating in each of the cell types. These efforts have been hampered because few transcription factor genes expressed in the female gametophyte have been identified. To identify such genes, we undertook a large-scale differential expression screen followed by promoter-fusion analysis to detect transcription-factor genes transcribed in the Arabidopsis female gametophyte. Results Using quantitative reverse-transcriptase PCR, we analyzed 1,482 Arabidopsis transcription-factor genes and identified 26 genes exhibiting reduced mRNA levels in determinate infertile 1 mutant ovaries, which lack female gametophytes, relative to ovaries containing female gametophytes. Spatial patterns of gene transcription within the mature female gametophyte were identified for 17 transcription-factor genes using promoter-fusion analysis. Of these, ten genes were predominantly expressed in a single cell type of the female gametophyte including the egg cell, central cell and the antipodal cells whereas the remaining seven genes were expressed in two or more cell types. After fertilization, 12 genes were transcriptionally active in the developing embryo and/or endosperm. Conclusions We have shown that our quantitative reverse-transcriptase PCR differential-expression screen is sufficiently sensitive to detect transcription-factor genes transcribed in the female gametophyte. Most of the genes identified in this study have not been reported previously as being expressed in the female gametophyte. Therefore, they might represent novel regulators and provide entry points for reverse genetic and molecular approaches to uncover the gene regulatory networks underlying female gametophyte development.Cellular and Molecular [email protected]

Gold substrate-induced single-mode lasing of GaN nanowires

We demonstrate a method for mode-selection by coupling a GaN nanowire laser to an underlying gold substrate. Multimode lasing of GaN nanowires is converted to single-mode behavior following placement onto a gold film. A mode-dependent loss is generated by the absorbing substrate to suppress multiple transverse-mode operation with a concomitant increase in lasing threshold of only ∼13%. This method provides greater flexibility in realizing practical single-mode nanowire lasers and offers insight into the design of metal-contacted nanoscale optoelectronics

Spatio-temporal expression patterns of Arabidopsis thaliana and Medicago truncatula defensin-like genes

Plant genomes contain several hundred defensin-like (DEFL) genes that encode short cysteine-rich proteins resembling defensins, which are well known antimicrobial polypeptides. Little is known about the expression patterns or functions of many DEFLs because most were discovered recently and hence are not well represented on standard microarrays. We designed a custom Affymetrix chip consisting of probe sets for 317 and 684 DEFLs from Arabidopsis thaliana and Medicago truncatula, respectively for cataloging DEFL expression in a variety of plant organs at different developmental stages and during symbiotic and pathogenic associations. The microarray analysis provided evidence for the transcription of 71% and 90% of the DEFLs identified in Arabidopsis and Medicago, respectively, including many of the recently annotated DEFL genes that previously lacked expression information. Both model plants contain a subset of DEFLs specifically expressed in seeds or fruits. A few DEFLs, including some plant defensins, were significantly up-regulated in Arabidopsis leaves inoculated with Alternaria brassicicola or Pseudomonas syringae pathogens. Among these, some were dependent on jasmonic acid signaling or were associated with specific types of immune responses. There were notable differences in DEFL gene expression patterns between Arabidopsis and Medicago, as the majority of Arabidopsis DEFLs were expressed in inflorescences, while only a few exhibited root-enhanced expression. By contrast, Medicago DEFLs were most prominently expressed in nitrogen-fixing root nodules. Thus, our data document salient differences in DEFL temporal and spatial expression between Arabidopsis and Medicago, suggesting distinct signaling routes and distinct roles for these proteins in the two plant species

Nascent Focal Adhesions Are Responsible for the Generation of Strong Propulsive Forces in Migrating Fibroblasts

Fibroblast migration involves complex mechanical interactions with the underlying substrate. Although tight substrate contact at focal adhesions has been studied for decades, the role of focal adhesions in force transduction remains unclear. To address this question, we have mapped traction stress generated by fibroblasts expressing green fluorescent protein (GFP)-zyxin. Surprisingly, the overall distribution of focal adhesions only partially resembles the distribution of traction stress. In addition, detailed analysis reveals that the faint, small adhesions near the leading edge transmit strong propulsive tractions, whereas large, bright, mature focal adhesions exert weaker forces. This inverse relationship is unique to the leading edge of motile cells, and is not observed in the trailing edge or in stationary cells. Furthermore, time-lapse analysis indicates that traction forces decrease soon after the appearance of focal adhesions, whereas the size and zyxin concentration increase. As focal adhesions mature, changes in structure, protein content, or phosphorylation may cause the focal adhesion to change its function from the transmission of strong propulsive forces, to a passive anchorage device for maintaining a spread cell morphology

Reactive Oxygen Species Generation in Human Cells by a Novel Magnetic Resonance Imaging Contrast Agent

The novel positive-contrast magnetic resonance imaging (MRI) marker C4 consists of an aqueous solution of cobalt chloride (CoCl 2 ) complexed with the chelator N-acetylcysteine (NAC). We evaluated whether the presence of C4 or its components would produce reactive oxygen species (ROS, including hydroxyl, peroxyl, or other reactive oxygen species) in cultured cells. Human cancer or normal cells were incubated with 1% (w/v) CoCl 2 ·6H 2 O or 2% NAC or a combination of both (1% CoCl 2 ·6H 2 O: 2% NAC in an aqueous solution, abbreviated as Co: NAC) in the presence or absence of H 2 O 2 . Intracellular ROS levels were measured and quantified by change in relative fluorescence units. Student\u27s t-tests were used. In all cell lines exposed to 1000 μM H 2 O 2 , the Co: NAC led to ≥94.7% suppression of ROS at 5 minutes and completely suppressed ROS at 60 and 90 minutes; NAC suppressed ROS by ≥76.6% at 5 minutes and by ≥94.5% at 90 minutes; and CoCl 2 ·6H 2 O suppressed ROS by ≥37.2% at 30 minutes and by ≥48.6% at 90 minutes. These results demonstrate that neither Co: NAC nor its components generated ROS; rather, they suppressed ROS production in cultured cells, suggesting that C4 would not enhance ROS production in clinical use