Editing Function Research Articles

MORF9 increases the RNA-binding activity of PLS-type pentatricopeptide repeat protein in plastid RNA editing.

RNA editing is a post-transcriptional process that modifies the genetic information on RNA molecules. In flowering plants, RNA editing usually alters cytidine to uridine in plastids and mitochondria. The PLS-type pentatricopeptide repeat (PPR) protein and the multiple organellar RNA editing factor (MORF, also known as RNA editing factor interacting protein (RIP)) are two types of key trans-acting factors involved in this process. However, how they cooperate with one another remains unclear. Here, we have characterized the interactions between a designer PLS-type PPR protein (PLS)3PPR and MORF9, and found that RNA-binding activity of (PLS)3PPR is drastically increased on MORF9 binding. We also determined the crystal structures of (PLS)3PPR, MORF9 and the (PLS)3PPR-MORF9 complex. MORF9 binding induces significant compressed conformational changes of (PLS)3PPR, revealing the molecular mechanisms by which MORF9-bound (PLS)3PPR has increased RNA-binding activity. Similarly, increased RNA-binding activity is observed for the natural PLS-type PPR protein, LPA66, in the presence of MORF9. These findings significantly expand our understanding of MORF function in plant organellar RNA editing.

Loggerhead sea turtle embryos (Caretta caretta) regulate expression of stress response and developmental genes when exposed to a biologically realistic heat stress.

Oviparous reptile embryos are expected to breach their critical thermal maxima if temperatures reach those predicted under current climate change models due to the lack of the maternal buffering processes and parental care. Heat-shock proteins (HSPs) are integral in the molecular response to thermal stress, and their expression is heritable, but the roles of other candidate families such as the heat-shock factors (HSFs) have not been determined in reptiles. Here, we subject embryonic sea turtles (Caretta caretta) to a biologically realistic thermal stress and employ de novo transcriptomic profiling of brain tissue to investigate the underlying molecular response. From a reference transcriptome of 302293 transcripts, 179 were identified as differentially expressed between treatments. As anticipated, genes enriched in the heat-shock treatment were primarily associated with the Hsp families, or were genes whose products play similar protein editing and chaperone functions (e.g. bag3, MYOC and serpinh1). Unexpectedly, genes encoding the HSFs were not significantly upregulated under thermal stress, indicating their presence in unstressed cells in an inactive state. Genes that were downregulated under thermal stress were less well functionally defined but were associated with stress response, development and cellular organization, suggesting that developmental processes may be compromised at realistically high temperatures. These results confirm that genes from the Hsp families play vital roles in the thermal tolerance of developing reptile embryos and, in addition with a number of other genes, should be targets for evaluating the capacity of oviparous reptiles to respond adaptively to the effects of climate change.

Enhancing the Catalytic Deamination Activity of APOBEC3C Is Insufficient to Inhibit Vif-Deficient HIV-1

The retroviral restriction factors of the APOBEC3 (A3) cytidine deaminase family catalyze the deamination of cytidines in single-stranded viral DNA. APOBEC3C (A3C) is a strong antiviral factor against viral infectivity factor (vif)-deficient simian immunodeficiency virus Δvif, which is, however, a weak inhibitor against human immunodeficiency virus (HIV)-1 for reasons unknown. The precise link between the antiretroviral effect of A3C and its catalytic activity is incompletely understood. Here, we show that the S61P mutation in human A3C (A3C.S61P) boosted hypermutation in the viral genomes of simian immunodeficiency virus Δvif and murine leukemia virus but not in human immunodeficiency virus HIV-1Δvif. The enhanced antiviral activity of A3C.S61P correlated with enhanced in vitro cytidine deamination. Furthermore, the S61P mutation did not change the substrate specificity of A3C, ribonucleoprotein complex formation, self-association, Zinc coordination, or viral incorporation features. We propose that local structural changes induced by the serine-to-proline substitution are responsible for the gain of catalytic activity of A3C.S61P. Our results are a first step toward an understanding of A3C's DNA binding capacity, deamination-dependent editing, and antiviral functions at the molecular level. We conclude that the enhanced enzymatic activity of A3C is insufficient to restrict HIV-1, indicating an unknown escape mechanism of HIV-1.

Hypervideos and Interactive Multimedia Presentations

Hypervideos and interactive multimedia presentations allow the creation of fully interactive and enriched video. It is possible to organize video scenes in a nonlinear way. Additional information can be added to the video ranging from short descriptions to images and more videos. Hypervideos are video-based but also provide navigation between video scenes and additional multimedia elements. Interactive multimedia presentations consist of different media with a temporal and spatial synchronization that can be navigated via hyperlinks. Their creation and description requires description formats, multimedia models, and standards—as well as players. Specialized authoring tools with advanced editing functions allow authors to manage all media files, link and arrange them to an overall presentation, and keep an overview during the whole process. They considerably simplify the creation process compared to writing and editing description documents in simple text editors. Data formats need features that describe interactivity and nonlinear navigation while maintaining temporal and spatial synchronization. Players should be easy to use with extended feature sets keeping elements synchronized. In this article, we analyzed more than 400 papers for relevant work in this field. From the findings we discovered a set of trends and unsolved problems, and propose directions for future research.

A Single-Target Mitochondrial RNA Editing Factor of Funaria hygrometrica Can Fully Reconstitute RNA Editing at Two Sites in Physcomitrella patens.

Nuclear-encoded pentatricopeptide repeat (PPR) proteins are key factors for site-specific RNA editing, converting cytidines into uridines in plant mitochondria and chloroplasts. All editing factors in the model moss Physcomitrella patens have a C-terminal DYW domain with similarity to cytidine deaminase. However, numerous editing factors in flowering plants lack such a terminal DYW domain, questioning its immediate role in the pyrimidine base conversion process. Here we further investigate the Physcomitrella DYW-type PPR protein PPR_78, responsible for mitochondrial editing sites cox1eU755SL and rps14eU137SL. Complementation assays with truncated proteins demonstrate that the DYW domain is essential for full PPR_78 editing functionality. The DYW domain can be replaced, however, with its counterpart from another editing factor, PPR_79. The PPR_78 ortholog of the related moss Funaria hygrometrica fully complements the Physcomitrella mutant for editing at both sites, although the editing site in rps14 is lacking in Funaria. Editing factor orthologs in different taxa may thus retain editing capacity for multiple sites despite the absence of editing requirement.

Homocysteine Editing, Thioester Chemistry, Coenzyme A, and the Origin of Coded Peptide Synthesis †.

Aminoacyl-tRNA synthetases (AARSs) have evolved “quality control” mechanisms which prevent tRNA aminoacylation with non-protein amino acids, such as homocysteine, homoserine, and ornithine, and thus their access to the Genetic Code. Of the ten AARSs that possess editing function, five edit homocysteine: Class I MetRS, ValRS, IleRS, LeuRS, and Class II LysRS. Studies of their editing function reveal that catalytic modules of these AARSs have a thiol-binding site that confers the ability to catalyze the aminoacylation of coenzyme A, pantetheine, and other thiols. Other AARSs also catalyze aminoacyl-thioester synthesis. Amino acid selectivity of AARSs in the aminoacyl thioesters formation reaction is relaxed, characteristic of primitive amino acid activation systems that may have originated in the Thioester World. With homocysteine and cysteine as thiol substrates, AARSs support peptide bond synthesis. Evolutionary origin of these activities is revealed by genomic comparisons, which show that AARSs are structurally related to proteins involved in coenzyme A/sulfur metabolism and non-coded peptide bond synthesis. These findings suggest that the extant AARSs descended from ancestral forms that were involved in non-coded Thioester-dependent peptide synthesis, functionally similar to the present-day non-ribosomal peptide synthetases.

Associating Physical Activity Levels with Motor Performance and Physical Function in Childhood Survivors of Acute Lymphoblastic Leukemia.

Purpose: This cross-sectional, observational study investigated whether physical activity (PA) levels are associated with motor performance and physical function in children after treatment for acute lymphoblastic leukemia (ALL). Method: Participants aged 8-13 years who had completed treatment for ALL (3-36 months post-treatment) were tested at their oncology long-term follow-up appointment at the British Columbia Children's Hospital. PA level was measured using the Physical Activity Questionnaire for Older Children (PAQ-C). Motor performance was measured using the Bruininks-Oseretsky Test of Motor Proficiency, Second Edition, Short Form (BOT-2 SF), and physical function was measured using the 6-minute walk test (6MWT). Results: Thirteen children completed testing. PAQ-C scores were not associated with BOT-2 SF or 6MWT performance. Eleven children (85%) performed below the norm for the 6MWT. Children with elevated body mass index had poorer 6MWT but similar PAQ-C scores. Conclusion: PA was not found to be associated with motor performance and physical function. Participants who were overweight or obese had poorer 6MWT performance, which may indicate the need for closer monitoring of post-treatment weight status and physical function in the oncology follow-up setting.

Quality Control by Isoleucyl-tRNA Synthetase of Bacillus subtilis Is Required for Efficient Sporulation

Isoleucyl-tRNA synthetase (IleRS) is an aminoacyl-tRNA synthetase whose essential function is to aminoacylate tRNAIle with isoleucine. Like some other aminoacyl-tRNA synthetases, IleRS can mischarge tRNAIle and correct this misacylation through a separate post-transfer editing function. To explore the biological significance of this editing function, we created a ileS(T233P) mutant of Bacillus subtilis that allows tRNAIle mischarging while retaining wild-type Ile-tRNAIle synthesis activity. As seen in other species defective for aminoacylation quality control, the growth rate of the ileS(T233P) strain was not significantly different from wild-type. When the ileS(T233P) strain was assessed for its ability to promote distinct phenotypes in response to starvation, the ileS(T233P) strain was observed to exhibit a significant defect in formation of environmentally resistant spores. The sporulation defect ranged from 3-fold to 30-fold and was due to a delay in activation of early sporulation genes. The loss of aminoacylation quality control in the ileS(T233P) strain resulted in the inability to compete with a wild-type strain under selective conditions that required sporulation. These data show that the quality control function of IleRS is required in B. subtilis for efficient sporulation and suggests that editing by aminoacyl-tRNA synthetases may be important for survival under starvation/nutrient limitation conditions.

The pentatricopeptide repeat protein EMP9 is required for mitochondrial ccmB and rps4 transcript editing, mitochondrial complex biogenesis and seed development in maize.

Pentatricopeptide repeat (PPR) proteins comprise a large family of sequence-specific RNA binding proteins in land plants. Because of its large family size and frequent embryo lethality in the mutants, molecular functions and physiological roles of many PPR proteins are unknown. Through characterization of an empty pericarp9 (emp9) mutant in maize (Zea mays), we defined the functions of EMP9 in mitochondrial RNA editing, respiratory complex formation and seed development. Mu insertions in different regions of Emp9 facilitated dissection of the domain functions of the EMP9. Through genetic and functional analyses of multiple alleles, we showed that deletions of two N-terminal PPR motifs and partial E+ domain do not eliminate the editing function of EMP9. Emp9 encodes an E+ subclass PPR protein that is localized in mitochondria. Loss of EMP9 function abolishes the C-to-U editing of ccmB-43 and rps4-335 sites in mitochondria. The loss of editing at ccmB-43 and rps4-335 affects the maturation of cytochrome c and impairs the biogenesis of mitochondrial respiratory complexes, particularly complex III. This work extends our understanding of PPR-E+ protein in editing function and seed development, and provides insights into the molecular function of mitochondrial CcmB protein in higher plants.

Discovery and Investigation of Natural Editing Function against Artificial Amino Acids in Protein Translation.

Fluorine being not substantially present in the chemistry of living beings is an attractive element in tailoring novel chemical, biophysical, and pharmacokinetic properties of peptides and proteins. The hallmark of ribosome-mediated artificial amino acid incorporation into peptides and proteins is a broad substrate tolerance, which is assumed to rely on the absence of evolutionary pressure for efficient editing of artificial amino acids. We used the well-characterized editing proficient isoleucyl-tRNA synthetase (IleRS) from Escherichia coli to investigate the crosstalk of aminoacylation and editing activities against fluorinated amino acids. We show that translation of trifluoroethylglycine (TfeGly) into proteins is prevented by hydrolysis of TfeGly-tRNAIle in the IleRS post-transfer editing domain. The remarkable observation is that dissociation of TfeGly-tRNAIle from IleRS is significantly slowed down. This finding is in sharp contrast to natural editing reactions by tRNA synthetases wherein fast editing rates for the noncognate substrates are essential to outcompete fast aa-tRNA dissociation rates. Using a post-transfer editing deficient mutant of IleRS (IleRSAla10), we were able to achieve ribosomal incorporation of TfeGly in vivo. Our work expands the knowledge of ribosome-mediated artificial amino acid translation with detailed analysis of natural editing function against an artificial amino acid providing an impulse for further systematic investigations and engineering of the translation and editing of unusual amino acids.

The Anti-HIV-1 Cytidine Deaminase APOBEC3G Is a Cellular Site-Specific RNA Editing Enzyme

Background:The APOBEC3 (A3) family of cytidine deaminases in primates is comprised of seven homologous enzymes that are structurally related to the RNA editing enzyme APOBEC1. APOBEC3G (A3G) is a restriction factor for HIV-1 and endogenous retroviruses, and is highly expressed in T lymphocytes. Encapsidation of A3G into HIV-1 particles is essential for its antiviral activity which leads to hypermutation of its cDNA in target cells, and requires RNA binding by A3G to form a ribonucleoprotein complex with viral proteins. A3G can also reduce HIV-1 production in producer cells independently of its DNA deaminating activity. A3G has homologous N-and C-terminal catalytic domains (NTD and CTD) but only the CTD is active for deamination of ssDNAs. The zinc-coordinating catalytic residues as well as non-catalytic residues in A3G-NTD are known to bind RNA and this interaction is required for A3G's binding to the HIV-1 nucleocapsid for recruitment into nascent virions as well as for A3G dimerization. A3G binds to DNA and RNA substrates with similar affinity. Thus far, studies have demonstrated DNA deamination by A3G whereas deamination has not been observed in HIV-1 RNA or synthetic RNA oligonucleotides, thereby, ruling out the RNA editing function of A3G. We recently described that the structurally related enzyme A3A induces widespread site-specific C-to-U RNA editing of cellular transcripts in pro-inflammatory macrophages and in monocytes exposed to hypoxia and/or interferons. We hypothesized that A3G may also have RNA editing function, which may play a role in HIV-1 restriction.Methods:To determine if A3G is capable of RNA editing, we transiently overexpressed the protein in 293T cells, a model routinely used by various labs to study A3G function and its mode of HIV-1 restriction, and then performed transcriptome-wide RNA sequencing (RNA-Seq), Sanger sequencing and site-directed mutagenesis.Results: RNA sequencing analysis showed site-specific RNA editing in hundreds of genes' transcripts, including approximately 200 genes that acquire protein recoding. The transcripts edited by A3G are largely distinct from those edited by A3A. We find that several host genes including NMT1, CHMP4B, MAPK1, ACIN1, MED1, NFAT5, RBM14 which areinvolved in HIV-1 infection acquire pathogenic recoding RNA mutations by A3G-mediated RNA editing. By performing Sanger sequencing of PCR-amplified cDNA, we validated site-specific, non-synonymous C-to-U RNA editing for 21 of 21 (100%) tested sites in 20 genes that we had selected for experimental confirmation. As expected no genomic mutations were seen in the DNA sequences corresponding to the RNA-edited sites in 11 tested genes. The discovery of A3G's RNA editing function prompted us to study the role of the N-terminal domain in RNA editing. We made mutations in the zinc-coordinating and non-catalytic residues in both N-terminal and C-terminal domains of A3G. We demonstrate that mutating zinc-coordinating residues in either N- and C-terminal domains of A3G in 293T cells greatly reduce or abolish editing in its target transcripts.Conclusions: We demonstrate a novel RNA editing function for the A3G cytidine deaminase. Our study shows that the RNAs of genes involved in HIV-1 replication, assembly, transcription and infectivity are targets of A3G-mediated RNA editing. This result raises the possibility that the editing of host transcripts may be a novel mechanism by which HIV-1 infection is inhibited by A3G. Our findings suggest a previously unrecognized role for the N-terminal domain of A3G in RNA editing. A3G is the second of the seven members of the APOBEC3 family of cytidine deaminases and the first two-domain cytidine deaminase for which a previously unrecognized RNA editing activity has been discovered. It suggests that other APOBEC3 proteins may also possess hitherto unknown RNA editing activity that may underpin some of their biological roles. Our findings have the potential to significantly expand on the role of C-to-U RNA editing in epitranscriptomic regulation in T lymphocytes, define specific gene targets of A3G-mediated RNA editing and open new avenues of inquiry on the functions of APOBEC3 genes in HIV-1 restriction. DisclosuresNo relevant conflicts of interest to declare.

The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme.

APOBEC3G is a cytidine deaminase with two homologous domains and restricts retroelements and HIV-1. APOBEC3G deaminates single-stranded DNAs via its C-terminal domain, whereas the N-terminal domain is considered non-catalytic. Although APOBEC3G is known to bind RNAs, APOBEC3G-mediated RNA editing has not been observed. We recently discovered RNA editing by the single-domain enzyme APOBEC3A in innate immune cells. To determine if APOBEC3G is capable of RNA editing, we transiently expressed APOBEC3G in the HEK293T cell line and performed transcriptome-wide RNA sequencing. We show that APOBEC3G causes site-specific C-to-U editing of mRNAs from over 600 genes. The edited cytidines are often flanked by inverted repeats, but are largely distinct from those deaminated by APOBEC3A. We verified protein-recoding RNA editing of selected genes including several that are known to be involved in HIV-1 infectivity. APOBEC3G co-purifies with highly edited mRNA substrates. We find that conserved catalytic residues in both cytidine deaminase domains are required for RNA editing. Our findings demonstrate the novel RNA editing function of APOBEC3G and suggest a role for the N-terminal domain in RNA editing.

Heat Increases the Editing Efficiency of Human Papillomavirus E2 Gene by Inducing Upregulation of APOBEC3A and 3G

Apolipoprotein B mRNA-editing catalytic polypeptide (APOBEC) 3 proteins have been identified as potent viral DNA mutators and have broad antiviral activity. In this study, we demonstrated that apolipoprotein B mRNA-editing catalytic polypeptide 3A (A3A) and A3G expression levels were significantly upregulated in human papillomavirus (HPV)-infected cell lines and tissues. Heat treatment resulted in elevated expression of A3A and A3G in a temperature-dependent manner in HPV-infected cells. Correspondingly, HPV-infected cells heat-treated at 44 °C showed accumulated G-to-A or C-to-T mutation in HPV E2 gene. Knockdown of A3A or A3G could promote cell viability, along with the lower frequency of A/T in HPV E2 gene. In addition, regressing genital viral warts also harbored high G-to-A or C-to-T mutation in HPV E2 gene. Taken together, we demonstrate that apolipoprotein B mRNA-editing catalytic polypeptide 3 expression and editing function was heat sensitive to a certain degree, partly explaining the mechanism of action of local hyperthermia to treat viral warts.

Development of the Story Template Design and Digital Contents for Promoting Young Children’s Creativity

Background/Objectives: Although various teaching, learning methods have been developed to utilize computers and ICTs for young children’s creativity education so far, limitations that do not satisfy learner’s changing needs and tastes, and do not utilize the most recent information technologies, still exist. Methods/Statistical Analysis: First, we provided young children with well-known fairy-tale scenarios or scenarios with good cinematic quality, and customized story templates, in which young children may freely and creatively reconfigure each frame of the story by using editing functions. Second, we designed templates to provide young children with subjects indirectly and directly to create an entirely new story. The proposed procedures for developing digital contents are designed to collectively perform young children-directed experiences and plays and to gain young children’s attention by utilizing images, videos, and Sound Effects (SE). Next, we developed creative scenarios that focus on developing young children’s creativity and vocabulary by configuring contents for age 3-5 that reflect traits of young children development. Then, based on existing fairy tales, we provided templates including characters, backgrounds, overall scenarios etcetera, which teachers use in classes. Therefore, the templates are designed young children to modify and reconfigure the given story. Finally, we adjusted quantities of digital contents by focusing on highlights of the template, and configured the contents by sentences. We also considered a linkage between elementary curriculum and 0-2 national child-care curriculum, to configure the templates. In addition, we created the templates for young children to freely edit and modify. Findings: Digital contents based on the story template have utilized the 3 dimensional simulations, rather than existing 2 dimensional texture mapping approaches, to provide an environment that enables young children to freely reconfigure and modify stories by using various editing functions such as size and motion changes. Digital contents based on the proposed story template also enable young children to creatively reconfigure each component of the given story, thereby learning more vocabulary, structuring finer sentence, conceiving the subject of the story, and enhancing creativity and originality. Improvements/Applications: The digital contents that use the proposed story templates lead young children to infinite reminiscent of action sequences, serve as a source of originality that enables magnificent thoughts. Through this imagination, creativity of young children shall be developed.

Evaluating esthetics for user-sketched layouts of clustered graphs with known clustering information

This paper aims to empirically analyze the esthetics for user-sketched layouts of clustered graphs with known clustering information. In our experiments, given not only the adjacency list of a clustered graph but also its predefined clustering information, each participant was asked to manually sketch clustered graphs “nicely” from scratch on a tablet system using a stylus. Different from previous works, the main concern in this paper is on which graph drawing esthetics people favor when sketching their own drawings of clustered graphs with known clustering information. Another concern of this paper is on the esthetics of clustered graph layouts employed by participants which include not only characteristics and structures of the final graph layouts but also the behavior of user's sketching process (including layout creation and adjustment). By observing all layouts and drawing processes, the drawing strategies which participants applied and the drawing esthetics are analyzed. Results show that most participants were unsurprisingly able to draw graphs with clear presence of bridge edges and clustering cohesiveness; more importantly, to distinguish clusters within the restricted-size tablet screen during the drawing process, some of the participants were still able to make each cluster with fewer edge crossings, more symmetries, and more alignment of grid in a smaller drawing area where the cluster spreads. Our results support that to alleviate user's complex drawing tasks, aside from the grid-based editing function suggested by the previous work, graph drawing systems should also provide the clustering information if the structure of the graph to be drawn is known.

The Form and Function of Dialectical Cinécriture in Beckett's Company

In this essay I argue that throughout Company Beckett adopts the technique of dialectical montage, which he encountered in the work of Eisenstein, to create a radically new autographical form of transmedial cinécriture. I suggest that this cinécriture enables Beckett not only intellectually to communicate the provisionality of self to readers, but also bodily to engage readers in a dialectical dynamic by which they directly experience the self for what it really is: an always-provisional synthesis in an always-provisional time and space. I begin the essay by considering the motivating factors that may have compelled Beckett to combine elements of film, radio, and prose in the late 1970s, after decades of resisting transmedial adaptations. I then examine the form and function of dialectical montage and constructive editing in Company. Next, I outline the form and function of intellectual montage and deep-focus space-time throughout the text and, in particular, in the penultimate watch sequence. Finally, I elaborate upon the ethical implications of the dialectic that these techniques set in motion.

A 'suicide' CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans.

Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This ‘suicide’ CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans.

Upper respiratory infection during pregnancy and neurodevelopmental outcomes among offspring

ObjectiveMaternal infection during pregnancy is associated with psychiatric disorders among offspring. The aim of this study was to investigate associations between upper respiratory infection (URI) in pregnancy and measures of cognitive and behavioral outcomes in child offspring. Materials and methodsA longitudinal study of 534 mother-child pairs with information regarding prenatal exposures collected through an interview conducted on average one year after delivery and subsequent participation in a childhood cognitive and psychosocial assessment between the ages 5–12years. Childhood cognition was measured using the Peabody Picture Vocabulary Test (PPVT-III) and the Beery-Buktenica Test of Visual Motor Integration-Fifth Edition (VMI-5) and behavioral function measured using the Child Behavior Checklist (CBCL) and teacher-report using the Teacher Report Form (TRF). Adjusted mean differences (adjMD) in outcome measures were calculated between mothers reporting the presence or absence of a URI during pregnancy. ResultsURI during pregnancy was not associated with the two measures of cognition given to offspring, but was associated with modest increases in total behavioral problems reported by mothers (adjMD: 3.72; CI: 1.91–5.54) and teachers (adjMD: 2.74; CI: 0.97–4.50). We observed differences in CBCL and TRF scores based on timing of URI: infections in mid-pregnancy (lunar months 4–5) were associated with poorer scores than were infections in early pregnancy (lunar months 2–3). ConclusionsIn general, URI in pregnancy was not associated with decrements in childhood cognition, but may be associated with behavior problems.

A redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum.

BackgroundA highly efficient CRISPR/Cas9-based marker-free genome editing system has been established in Plasmodium falciparum (Pf). However, with the current methods, two drug-selectable markers are needed for episome retention, which may present hurdles for consecutive genome manipulations due to the limited number of available selectable markers. The loading capacity of donor DNA is also unsatisfactory due to the large size of the Cas9 nuclease and sgRNA co-expression system, which limits the size of knock-in DNA fragments. Because of the inefficient end joining (EJ) DNA repair mechanism of Pf, a suicide-rescue approach could be used to address the challenges. Cas9 nuclease and sgRNA were co-expressed from a single plasmid (suicide vector) with one selectable marker, and the donor DNA was ligated into the other plasmid (rescue vector) containing only the ampicillin-resistance gene (AmpR) and a ColEl replication origin (ori). Nonetheless, whether this approach can mediate even the regular gene editing in Pf remains unknown. This study aimed to demonstrate the basic gene editing function of this Cas9-mediated suicide-rescue system.FindingsThe suicide and rescue vectors were constructed and co-transfected into Pf3D7. This system worked as expected when used to disrupt the Pfset2 gene and to insert a green fluorescent protein-renilla luciferase (gfp-ruc) fusion gene cassette of 3334 base pairs (bp) into the Pf47 locus, demonstrating that the suicide vector actually induced double-strand breaks (DSBs) and that the rescue vector functioned without maintenance via drug selection.ConclusionsThe adapted marker-free CRISPR/Cas9 system with only a single episome-selectable marker performs well as the current systems for general gene editing which lays a solid foundation for further studies including consecutive gene manipulations and large gene knock-ins.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1487-4) contains supplementary material, which is available to authorized users.

Histone chaperones FACT and Spt6 prevent histone variants from turning into histone deviants.

Histone variants are specialized histones which replace their canonical counterparts in specific nucleosomes. Together with histone post-translational modifications and DNA methylation, they contribute to the epigenome. Histone variants are incorporated at specific locations by the concerted action of histone chaperones and ATP-dependent chromatin remodelers. Recent studies have shown that the histone chaperone FACT plays key roles in preventing pervasive incorporation of two histone variants: H2A.Z and CenH3/CENP-A. In addition, Spt6, another histone chaperone, was also shown to be important for appropriate H2A.Z localization. FACT and Spt6 are both associated with elongating RNA polymerase II. Based on these two examples, we propose that the establishment and maintenance of histone variant genomic distributions depend on a transcription-coupled epigenome editing (or surveillance) function of histone chaperones.