Editing Function Research Articles

Aminoacyl-tRNA synthetases and the evolution of coded peptide synthesis: the Thioester World.

Coded peptide synthesis must have been preceded by a prebiotic stage, in which thioesters played key roles. Fossils of the Thioester World are found in extant aminoacyl-tRNA synthetases (AARSs). Indeed, studies of the editing function reveal that AARSs have a thiol-binding site in their catalytic modules. The thiol-binding site confers the ability to catalyze aminoacyl~coenzyme A thioester synthesis and peptide bond formation. Genomic comparisons show that AARSs are structurally related to proteins involved in sulfur and coenzyme A metabolisms and peptide bond synthesis. These findings point to the origin of the amino acid activation and peptide bond synthesis functions in the Thioester World and suggest that the present-day AARSs had originated from ancestral forms that were involved in noncoded thioester-dependent peptide synthesis.

The Flipped Classroom: Two Learning Modes that Foster Two Learning Outcomes

The study involved student teachers enrolled in early childhood teaching at a teacher training institute in Hong Kong Special Administrative Region. Seventy-four students participated in flipped classroom activities during their first semester of study. Students were told to learn from online videos related to using image editing software in their own time and pace prior to the next class. When they met in class, they were asked to apply their recently acquired editing knowledge to edit an image of their own choice related to the theme of their group project. At the end of the activity, students were asked to complete an online questionnaire. It was found that students had rated all five questions relating to generic skills highly, with self-study skills rated the highest. They particularly enjoyed the flexibility of learning on their own time and pace as a benefit of the flipped classroom. Data collected from students’ project pages show they had used average of 3.22 editing features for the theme images for their project. Most groups had inserted text followed by using the filter function. It is possible that these two functions are more noticeable than other editing functions. In conclusion, students were able to apply their self-learnt knowledge in a real-life situation and they had also developed their generic skills via the flipped classroom pedagogy.

Keeping CRISPR/Cas on-Target

CRISPR/Cas, a microbial adaptive immune system, has recently been reshaped as a versatile genome editing approach, endowing genome engineering with high efficiency and robustness. The DNA endonuclease Cas, a component of CRISPR system, is directed to specific target within genomes by guide RNA (gRNA) and performs gene editing function. However, the system is still in its infancy and facing enormous challenges such as off-target mutation. Lots of attempts have been made to overcome such off-targeting and proven to be effective. In this review we focused on recent progress of increasing the CRISPR specificity realized by rational design of gRNA and modification of Cas9 endonuclease. Meanwhile the methods to screen off-target mutation and their effects are also discussed. Comprehensive consideration and rational design to reduce off-target mutation and selection of effective screening assay will greatly facilitate to achieve successful CRISPR/Cas system mediated gene editing.

Metabolomic pathway visualization tool outsourcing editing function.

Recent rapid improvements of measuring instrument enables us to perform various omics studies to simultaneous profile multiple molecules, which provides a holistic view of various molecular interactions, such as signal transaction, protein interactions, and metabolic pathways. Metabolomics is recently emerged omics that can identify and quantify low weight metabolites usually defined as organic molecules whose size is <; 1500 Da. In comparison to the other omics, the development of software tools to deal with metabolomic data is not matured. Conventional pathway drawing and visualization tool provide tool-specific unique functions, however, such user interface requires users to learn the usage and prevention for the use of these tools. Here, we developed a more generic pathway visualization tool. This tool incorporate pathway data yielded by common drawing tools, e.g. MS PowerPoint, and visualize the quantified values on the pathways. The statistical results also can be overlaid on each metabolite. The developed tools facilitate the interpreting metabolomic data in pathway forms.

Based on WebGIS platform developing remote sensing information systems of forest canopy density inversion

With Landsa 8 OLI data, field investigation of the sample data and the ASTER GDEM elevation data as the foundation, this paper views Jiangle County, Sanming City, Fujian Province as the study area, and selects the stepwise-linear regression model, the backward-linear regression model and partial-least squares model to study the crown density distribution of the study area. On the ArcGIS Server for the. NET development （ADF） platform, this system combines with the spatial database technology, and sets up a distributed information system on forest crown density inversion which is based on B/S model, so that this system can carry out basic GIS operations, data query, the model factor calculation, forest canopy density model implementation, data editing and output functions, etc. Basically the system can meet the regional forest resources structure parameters analysis especially the unity of the crown density information management and processing requirements, and supports a platform for structural parameters, data normalization management and forest resources sharing services for Jiangle County, Sanming City.［Ch, 6 fig. 15 ref.］

Modulation of Aminoacylation and Editing Properties of Leucyl-tRNA Synthetase by a Conserved Structural Module

A conserved structural module following the KMSKS catalytic loop exhibits α-α-β-α topology in class Ia and Ib aminoacyl-tRNA synthetases. However, the function of this domain has received little attention. Here, we describe the effect this module has on the aminoacylation and editing capacities of leucyl-tRNA synthetases (LeuRSs) by characterizing the key residues from various species. Mutation of highly conserved basic residues on the third α-helix of this domain impairs the affinity of LeuRS for the anticodon stem of tRNA(Leu), which decreases both aminoacylation and editing activities. Two glycine residues on this α-helix contribute to flexibility, leucine activation, and editing of LeuRS from Escherichia coli (EcLeuRS). Acidic residues on the β-strand enhance the editing activity of EcLeuRS and sense the size of the tRNA(Leu) D-loop. Incorporation of these residues stimulates the tRNA-dependent editing activity of the chimeric minimalist enzyme Mycoplasma mobile LeuRS fused to the connective polypeptide 1 editing domain and leucine-specific domain from EcLeuRS. Together, these results reveal the stem contact-fold to be a functional as well as a structural linker between the catalytic site and the tRNA binding domain. Sequence comparison of the EcLeuRS stem contact-fold domain with editing-deficient enzymes suggests that key residues of this module have evolved an adaptive strategy to follow the editing functions of LeuRS.

Native Variants of the MRB1 Complex Exhibit Specialized Functions in Kinetoplastid RNA Editing.

Adaptation and survival of Trypanosoma brucei requires editing of mitochondrial mRNA by uridylate (U) insertion and deletion. Hundreds of small guide RNAs (gRNAs) direct the mRNA editing at over 3,000 sites. RNA editing is controlled during the life cycle but the regulation of substrate and stage specificity remains unknown. Editing progresses in the 3’ to 5’ direction along the pre-mRNA in blocks, each targeted by a unique gRNA. A critical editing factor is the mitochondrial RNA binding complex 1 (MRB1) that binds gRNA and transiently interacts with the catalytic RNA editing core complex (RECC). MRB1 is a large and dynamic complex that appears to be comprised of distinct but related subcomplexes (termed here MRBs). MRBs seem to share a ‘core’ complex of proteins but differ in the composition of the ‘variable’ proteins. Since some proteins associate transiently the MRBs remain imprecisely defined. MRB1 controls editing by unknown mechanisms, and the functional relevance of the different MRBs is unclear. We previously identified two distinct MRBs, and showed that they carry mRNAs that undergo editing. We proposed that editing takes place in the MRBs because MRBs stably associate with mRNA and gRNA but only transiently interact with RECC, which is RNA free. Here, we identify the first specialized functions in MRBs: 1) 3010-MRB is a major scaffold for RNA editing, and 2) REH2-MRB contains a critical trans-acting RNA helicase (REH2) that affects multiple steps of editing function in 3010-MRB. These trans effects of the REH2 include loading of unedited mRNA and editing in the first block and in subsequent blocks as editing progresses. REH2 binds its own MRB via RNA, and conserved domains in REH2 were critical for REH2 to associate with the RNA and protein components of its MRB. Importantly, REH2 associates with a ~30 kDa RNA-binding protein in a novel ~15S subcomplex in RNA-depleted mitochondria. We use these new results to update our model of MRB function and organization.

DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader.

The DNA replication machinery is an important target for antibiotic development for increasingly drug resistant bacteria including Mycobacterium tuberculosis1. While blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In E. coli, the proofreading subunit of the replisome, the ε-exonuclease, is essential for high fidelity DNA replication2; however, we find that it is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase, DnaE1, encodes a novel editing function that proofreads DNA replication, mediated by an intrinsic 3′-5′ exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by greater than 3,000 fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP-domain mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader.

Genomic analysis of ADAR1 binding and its involvement in multiple RNA processing pathways.

Adenosine deaminases acting on RNA (ADARs) are the primary factors underlying adenosine to inosine (A-to-I) editing in metazoans. Here we report the first global study of ADAR1-RNA interaction in human cells using CLIP-Seq. A large number of CLIP sites are observed in Alu repeats, consistent with ADAR1's function in RNA editing. Surprisingly, thousands of other CLIP sites are located in non-Alu regions, revealing functional and biophysical targets of ADAR1 in the regulation of alternative 3' UTR usage and miRNA biogenesis. We observe that binding of ADAR1 to 3' UTRs precludes binding by other factors, causing 3' UTR lengthening. Similarly, ADAR1 interacts with DROSHA and DGCR8 in the nucleus and possibly out-competes DGCR8 in primary miRNA binding, which enhances mature miRNA expression. These functions are dependent on ADAR1's editing activity, at least for a subset of targets. Our study unfolds a broad landscape of the functional roles of ADAR1.

Use of Parametric 3D Modelling - Tying Parameter Values to Spreadsheets at Designing Molds for Plastic Injection

The current mechanical engineering is inconceivable without the implementation of CAx systems in design and manufacturing process of individual components. The automotive industry is a clear evidence of how CAx systems affect the innovation cycle of its product - a car. The innovation cycle in automotive was reduced from 8-12 years to the current 4-6 years. Even in this short interval automakers make some small design modifications called a facelift. Development in the automotive industry, therefore, is closely related to news and functionality CAx systems. CAD systems at the turn of the millennium are characterized as parametric graphic systems with a history tree of product creation. Parametric design implemented into CAD systems makes the model variable and open to rapid change management. The history tree in turn enables rapid editing and modification of forming or editing functions.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide family in virus-related diseases

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide(APOBEC)family are a group of active antiviral proteins that can edit DNA and RNA sequences by deamidizing enzymes. The APOBEC-like 3(APOBEC3)family was firstly noticed for its inhibitory effect against human immunodeficiency virus type 1(HIV-1)infection. With the development of research, the APOBEC family have been found to play an important role in other viral infections closely related to human diseases, such as human T-cell lymphotropic virus(HTLV), hepatitis C virus(HCV), hepatitis B virus(HBV), human papilloma virus(HPV), herpes simplex virus(HSV), etc. In addition, the editing function of the APOBEC family contributes not only to viral genetic diversity and evolution, but also to tumorigenesis. Key words: Virus diseases; Apolipoproteins B; Peptides

A Place to Call Home: Bioengineering Pluripotential Stem Cell Cultures

Pluripotent stem cells (PSCs) have the power to revolutionize the future of cell-based therapies and regenerative medicine. However, stem/progenitor cell use in the clinical arsenal has been hampered by discrepancies resulting from stem cell engineering and expansion, as well as in their (mass) differentiation in culture. Moreover, the manner in which external conditions affect PSC and induced-pluripotent stem cell lineage establishment as well as maturation remains controversial. In this review, we examine novel methods of cell engineering and the role of reprogramming transcription factors in PSC development. In addition, we explore the effect of external environmental signals on PSC cultivation and differentiation by elucidating key components of the primordial stem cell microenvironment, the blastocyst. Furthermore, we assess the effects of hypoxic conditions on DNA editing, gene expression, and protein function in PSC self-renewal and growth. Finally, we speculate on the principal use of gap junction subunit expression as relevant biomarkers of PSC fate. Improving bioreactor design and pertinent cell biomarker classification could vastly enhance manufactured stem cell yield and quality, thereby increasing the potency and safety of therapeutic cells to be used in regenerative medicine.

Cytidine Deaminase Motifs within the DYW Domain of Two Pentatricopeptide Repeat-containing Proteins Are Required for Site-specific Chloroplast RNA Editing

In angiosperm organelles, cytidines are converted to uridines by a deamination reaction in the process termed RNA editing. The C targets of editing are recognized by members of the pentatricopeptide repeat (PPR) protein family. Although other members of the editosome have begun to be identified, the enzyme that catalyzes the C-U conversion is still unknown. The DYW motif at the C terminus of many PPR editing factors contains residues conserved with known cytidine deaminase active sites; however, some PPR editing factors lack a DYW motif. Furthermore, in many PPR-DYW editing factors, the truncation of the DYW motif does not affect editing efficiency, so the role of the DYW motif in RNA editing is unclear. Here, a chloroplast PPR-DYW editing factor, quintuple editing factor 1 (QED1), was shown to affect five different plastid editing sites, the greatest number of chloroplast C targets known to be affected by a single PPR protein. Loss of editing at the five sites resulted in stunted growth and accumulation of apparent photodamage. Adding a C-terminal protein tag to QED1 was found to severely inhibit editing function. QED1 and RARE1, another plastid PPR-DYW editing factor, were discovered to require their DYW motifs for efficient editing. To identify specific residues critical for editing, conserved deaminase residues in each PPR protein were mutagenized. The mutant PPR proteins, when expressed in qed1 or rare1 mutant protoplasts, could not complement the editing defect. Therefore, the DYW motif, and specifically, the deaminase residues, of QED1 and RARE1 are required for editing efficiency.

TEI4LdoD: Textual Encoding and Social Editing in Web 2.0 Environments

In this article we describe how textual encoding is used in our current project of constructing a digital archive of Fernando Pessoa’s Livro do Desassossego [LdoD]. Our model for virtualizing the authorial and editorial forms of this unfinished work aims to create a dynamic archive that is open to user interaction and collaboration. A brief introduction to the theoretical rationale of the archive is followed by a description of our technical solution for TEI XML encoding that is responsive to dynamic changes over time. With our software architecture proposal for processing TEI markup, the Collaborative Digital Archive of the Book of Disquiet will be able to instantiate the cooperative and social editing functionalities of Web 2.0 environments.

Evidence for multiple, distinct ADAR-containing complexes in Xenopus laevis.

ADAR (adenosine deaminase acting on RNA) is an RNA-editing enzyme present in most metazoans that converts adenosines in double-stranded RNA targets into inosines. Although the RNA targets of ADAR-mediated editing have been extensively cataloged, our understanding of the cellular function of such editing remains incomplete. We report that long, double-stranded RNA added to Xenopus laevis egg extract is incorporated into an ADAR-containing complex whose protein components resemble those of stress granules. This complex localizes to microtubules, as assayed by accumulation on meiotic spindles. We observe that the length of a double-stranded RNA influences its incorporation into the microtubule-localized complex. ADAR forms a similar complex with endogenous RNA, but the endogenous complex fails to localize to microtubules. In addition, we characterize the endogenous, ADAR-associated RNAs and discover that they are enriched for transcripts encoding transcriptional regulators, zinc-finger proteins, and components of the secretory pathway. Interestingly, association with ADAR correlates with previously reported translational repression in early embryonic development. This work demonstrates that ADAR is a component of two, distinct ribonucleoprotein complexes that contain different types of RNAs and exhibit diverse cellular localization patterns. Our findings offer new insight into the potential cellular functions of ADAR.

MOST: a software environment for constraint-based metabolic modeling and strain design.

MOST (metabolic optimization and simulation tool) is a software package that implements GDBB (genetic design through branch and bound) in an intuitive user-friendly interface with excel-like editing functionality, as well as implementing FBA (flux balance analysis), and supporting systems biology markup language and comma-separated values files. GDBB is currently the fastest algorithm for finding gene knockouts predicted by FBA to increase production of desired products, but GDBB has only been available on a command line interface, which is difficult to use for those without programming knowledge, until the release of MOST. MOST is distributed for free on the GNU General Public License. The software and full documentation are available at http://most.ccib.rutgers.edu/. dslun@rutgers.edu.

Design and Implementation of an Embedded PLC System with Applied-Information Technology

To overcome the shortcomings of external PLC (Programmable Logic Controller) and soft PLC being widely employed in switch control of CNC machine tool, an embedded PLC system was developed by integrating AVR single chip ATMEGA169, PC bus technique and dual port RAM. Both hardware circuit and software of the PLC system were designed and implemented. The software of Industrial PC was developed by using ladder-diagram IDE (Integrated Design Environment) based on the platform of Borland C++ Builder. In this environment, it can fulfill edit, compilation and communication function of ladder-diagram file. It also can monitor and diagnose PLC running status. This developed embedded PLC system has the characteristics of compact structure, high communication and reliability. It can effectively satisfy real-time control requirements of CNC machine tool.

C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia: gain or loss of function?

Purpose of reviewThe molecular mechanisms that underlie chromosome 9 open reading frame 72 (C9orf72)-associated amyotrophic lateral sclerosis and frontotemporal dementia are rapidly emerging. Two potential disease mechanisms have been postulated – gain or loss of function. We provide an overview of recent advances that support or oppose gain-of-function and loss-of-function mechanisms.Recent findingsSince the discovery that a noncoding repeat expansion in C9orf72 was responsible for chromosome 9-linked amyotrophic lateral sclerosis and frontotemporal dementia in 2011, a plethora of studies have investigated clinical, pathological and mechanistic aspects of the disease. Loss of function is supported by reduced levels of C9orf72 in patient brain and functional work, revealing a role of the C9orf72 protein in endocytic and autophagic pathways and motor function. Gain of function is supported by the presence in patient brain of both repeat RNA and protein aggregates. Repeat RNA aggregates termed RNA foci, a hallmark of noncoding repeat expansion diseases, have been shown to sequester proteins involved in RNA splicing, editing, nuclear export and nucleolar function. Repeat-associated non-ATG dependent translation gives rise to toxic dipeptide repeat proteins that form inclusions in patient tissue. Antisense oligonucleotides targeting C9orf72 have shown promise for combating gain-of-function toxicity.SummaryRapid progress is being made towards understanding this common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Overall, the weight of data currently sits in favour of gain of function as the most important disease mechanism, which has important implications for the development of effective and targeted therapies.

High-Level Expression of an Allo-MHC II Transgene Can Be Achieved in Mouse Hepatocytes, But Lack of Chaperones and Accessory Molecules May Prevent Tolerance Induction.

Background: Previous studies have shown induction of specific tolerance and long term survival of fully allogeneic cardiac grafts when donor MHC class II genes were transferred to recipient bone marrow prior to transplantation. In our published data, expression of donor MHC class I (Kb) in recipient liver induced tolerance to Kb-expressing skin grafts. Here, we investigate the effect of high level recombinant Adeno-Associated Virus (AAV)-mediated expression of MHC class II in heart transplant setting. Methods: We developed an efficient liver-specific vector encoding H-2IAd (rAAV-IAd). C57BL/6 (B6, H-2b) WT and Foxp3GFPmice were injected with varying doses (5x108-5x1011 vector genome copies, vgc) to produce expression of H-2IAd on hepatocytes. Expression of IAd and costimulatory molecules was assessed by FACS on isolated hepatocytes, whilst expression of Invariant chain (Ii) and H-2M was determined by RT-PCR. Fully-allogeneic hearts from DBA/2 (H-2d) were transplanted into C57BL/6 at d7 or d100 post-transduction. Results: High level expression of H-2IAd on hepatocytes of B6 mice with doses of 5x1010 or 5x1011 rAAV-IAdwas observed.Figure: No Caption available.Foxp3+ CD4+ T cells were increased in the livers of mice receiving 5x1011 rAAV-IAd with 2±0.76% in normal ctrl, 2.4± 0.6% for 5x1010 IAd injected mice and 7.7±2.2% for 5x1011 IAd injected mice (p<0.03).Yet, the survival rate of hearts grafted into rAAV-IAd treated B6 mice was not improved compared to grafts in normal mice (MST 8d and 7d respectively). Hepatocytes from IAd-injected mice did not express CD40, CD80, CD86 or PD-L1. In addition, RT-PCR revealed minimal expression of H2-M and Ii, suggesting an impaired function in peptide editing of expressed IAd. Conclusion: High level expression of MHCII IAd can be achieved in recipient liver using rAAV vectors. However, initiating an immune response and tolerance to grafts bearing IAd might require supplying H2-M and invariant chain (Ii) genes for biosynthesis of fully functional MHCII IAd from hepatocytes.

Development of the Monitoring Guidance System for Slope Engineering

The slope monitoring is an important part of the slope engineering. Through the reasonable monitoring, the engineers can get all kinds of information and the state of slope stability; provide scientific basis for the forecast about instability. Facing various methods and diverse information, the engineers can not consult books and make project efficiently. Based on the objective of safe construction, a database system including a comprehensive range of information about monitoring design, construction technology and equipment is built by achieving the design ideas of rapid and concise. By utilizing the development tools of Visual Basic and database platform of Access, the monitoring guidance system is developed with the consultation, editing, and analysis functions. The system provides a complete slope monitoring information platform to guide the engineering staff to quickly and accurately develop the design of slope monitoring.