Transgene-assisted genetic screen identifies rsd-6 and novel genes as key components of antiviral RNA interference in Caenorhabditis elegans

© 2018 American Society for Microbiology. RNA interference (RNAi) is a widespread antiviral mechanism triggered by virus-produced double-stranded RNAs (dsRNAs). In Caenorhabditis elegans, antiviral RNAi involves a RIG-I-like RNA helicase, termed DRH-1 (dicer related RNA helicase 1), that is not required for classical RNAi triggered by artificial dsRNA. Currently, whether antiviral RNAi in C. elegans involves novel factors that are dispensable for classical RNAi remains an open question. To address this question, we designed and carried out a genetic screen that aims to identify novel genes involved in worm antiviral RNAi. By introducing extra copies of known antiviral RNAi genes into the reporter worms, we managed to reject alleles derived from 4 known antiviral RNAi genes, including the DRH-1 coding gene, during the screen. Our genetic screen altogether identified 25 alleles, which were assigned to 11 candidate genes and 2 known antiviral RNAi genes through genetic complementation tests. Using a mapping-by-sequencing strategy, we identified one of the candidate genes as rsd-6, a gene that helps maintain genome integrity through an endogenous gene-silencing pathway but was not known to be required for antiviral RNAi. More importantly, we found that two of the candidate genes are required for antiviral RNAi targeting Orsay virus, a natural viral pathogen of C. elegans, but dispensable for classical RNAi. Since drh-1 is so far the only antiviral RNAi gene not required for classical RNAi, we believe that our genetic screen led to identification of novel worm genes that may target virus-specific features to function in RNAi

Telomere Recombination Accelerates Cellular Aging in Saccharomyces cerevisiae

Telomeres are nucleoprotein structures located at the linear ends of eukaryotic chromosomes. Telomere integrity is required for cell proliferation and survival. Although the vast majority of eukaryotic species use telomerase as a primary means for telomere maintenance, a few species can use recombination or retrotransposon-mediated maintenance pathways. Since Saccharomyces cerevisiae can use both telomerase and recombination to replicate telomeres, budding yeast provides a useful system with which to examine the evolutionary advantages of telomerase and recombination in preserving an organism or cell under natural selection. In this study, we examined the life span in telomerase-null, post-senescent type II survivors that have employed homologous recombination to replicate their telomeres. Type II recombination survivors stably maintained chromosomal integrity but exhibited a significantly reduced replicative life span. Normal patterns of cell morphology at the end of a replicative life span and aging-dependent sterility were observed in telomerase-null type II survivors, suggesting the type II survivors aged prematurely in a manner that is phenotypically consistent with that of wild-type senescent cells. The shortened life span of type II survivors was extended by calorie restriction or TOR1 deletion, but not by Fob1p inactivation or Sir2p over-expression. Intriguingly, rDNA recombination was decreased in type II survivors, indicating that the premature aging of type II survivors was not caused by an increase in extra-chromosomal rDNA circle accumulation. Reintroduction of telomerase activity immediately restored the replicative life span of type II survivors despite their heterogeneous telomeres. These results suggest that telomere recombination accelerates cellular aging in telomerase-null type II survivors and that telomerase is likely a superior telomere maintenance pathway in sustaining yeast replicative life span

Saccharomyces cerevisiae Est3p dimerizes in vitro and dimerization contributes to efficient telomere replication in vivo

In Saccharomyces cerevisiae at least five genes, EST1, EST2, EST3, TLC1 and CDC13, are required for telomerase activity in vivo. The telomerase catalytic subunit Est2p and telomerase RNA subunit Tlc1 constitute the telomerase core enzyme. Est1p and Est3p are the other subunits of telomerase holoenzyme. In order to dissect the function of Est3p in telomere replication, we over-expressed and purified recombinant wild-type and mutant Est3 proteins. The wild-type protein, as well as the K71A, E104A and T115A mutants were able to dimerize in vitro, while the Est3p-D49A, -K68A or -D166A mutant showed reduced ability to dimerize. Mutations in Est3p that decreased dimerization also appeared to cause telomere shortening in vivo. Double point mutation of Est3p-D49A-K68A and single point mutation of Est3p-K68A showed similar telomere shortening, suggesting that the K68 residue might be more important for telomerase activity. The ectopic co-expression of K71A or T115A mutant with wild-type Est3p using centromere plasmids caused telomere shortening, while co-expression of the D49A, K68A, D86A or F103A mutants with wild-type Est3p had no effect on telomere length regulation. These data suggested that dimerization is important for Est3p function in vivo

Adaptive NN Control for Multisteering Plane Aircraft with Dead Zone or Backlash Input Nonlinearity

Considering that many factors such as actuator input dead zone, backlash, and external disturbance could affect the exactness of trajectory tracking, therewith a robust adaptive neural network control scheme on the basis of control allocation is proposed for the sake of tracking control of multisteering plane aircraft with actuator input dead zone or backlash nonlinearity. First of all, an actuator input dead zone or backlash nonlinearity control assignment model is established and the control allocation equation is derived. Secondly, the system nonlinear uncertainty is compensated by means of radial basis function neural network, and a robust term is introduced to achieve robustness against external disturbance and system errors. Finally, by utilizing Lyapunov stability theorem, it has been proved that all the signals in the closed-loop system are bounded, and the tracking error converges to a small residual set asymptotically. Simulation results on ICE101 multisteering plane aircraft demonstrate the outstanding tracking performance and strong robustness as well as effectiveness of the proposed approach, which can effectively overcome the adverse influence of dead zone, backlash nonlinearity, and external disturbance on the system

In silico screening of potentially bioactive-anti-functional dyspepsia constituents of Magnoliae officinalis Cortex based on molecular docking and network pharmacology

Purpose: To screen for bioactive anti-functional dyspepsia compounds from Magnoliae officinalis Cortex (Hou Po) and to identify the mechanism(s) of action involved.Methods: The compounds of Hou Po were collected from the literature. The related target proteins were identified from DrugBank. Through  “Libdock” module of Discovery Studio 3.5, the compounds were matched with related target proteins. Taking the Libdock score of the original ligand with target protein as standard, components with higher scores than this standard were considered as potential bioactive compounds. Based on Cytoscape software, the interaction networks of the bioactive compound-target protein complexes were mapped. On the other hand, the online DAVID database was used to analyze the GO enrichment and KEGG pathway of each target.Results: A total of 199 chemical constituents and 13 correlated target proteins were obtained. One hundred and thirty-nine (139) potential bioactive constituents were acquired based on molecular docking. Thirty-one (31) bioactive compounds were selected based on degree values in networkanalysis. “Palmitone” and “magnolignan G” which had the highest degree values were considered promising and leading compounds. The result of gene enrichment analysis showed that the bioactive compounds exerted their effects mainly via “neuroactive ligand-receptor interaction” pathway and “Cholinergic synapse” pathways.Conclusion: Based on molecular docking and network pharmacology technique, the material basis for the use of Hou Po in the treatment of FD has been revealed. This finding provides a useful guide in the development of Hou Po-based anti-FD drugs. Keywords: Magnolia officinalis, Hou Po, Molecular docking, Functional dyspepsia, Network pharmacolog

Neural Correlates of Orthographic Access in Mandarin Chinese Writing: An fMRI Study of the Word-Frequency Effect

Writing is an essential tool for human communication and involves multiple linguistic, cognitive, and motor processes. Chinese, a logographic writing system, differs remarkably from the writing systems of alphabetic languages. The neural substrates of Chinese writing are largely unknown. Using functional magnetic resonance imaging (fMRI) in a copying task, this study probed the neural underpinnings of orthographic access during Mandarin Chinese writing by employing the word-frequency effect. The results showed that writing low-frequency characters evoked greater activation in the bilateral superior/middle/inferior frontal gyrus, superior/inferior parietal lobule, and fusiform gyrus than writing high-frequency characters. Moreover, psychophysiological interaction (PPI) analysis demonstrated that the word-frequency effect modulated functional connectivity within the frontal-occipital networks and the parietal-occipital networks. Together, these findings illustrate the neural correlates of orthographic access for Mandarin Chinese writing, shedding new light on the cognitive architecture of writing across various writing systems

Curcumin reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia cell lines and primary sorted CD34+ acute myeloid leukemia cells

<p>Abstract</p> <p>Background</p> <p>Acute myeloid leukemia (AML) is an immunophenotypically heterogenous malignant disease, in which CD34 positivity is associated with poor prognosis. CD34⁺AML cells are 10-15-fold more resistant to daunorubicin (DNR) than CD34^-AML cells. Curcumin is a major component of turmeric that has shown cytotoxic activity in multiple cancers; however, its anti-cancer activity has not been well studied in DNR-insensitive CD34⁺AML cells. The aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34⁺AML cell lines (KG1a, Kasumi-1), DNR-sensitive U937 AML cells, and primary CD34⁺AML bone-marrow-derived cells.</p> <p>Methods</p> <p>Primary human CD34⁺cells were isolated from peripheral blood mononuclear cells or bone marrow mononuclear cells using a CD34 MicroBead kit. The growth inhibitory effects of curcumin were evaluated by MTT and colony-formation assays. Cell cycle distribution was examined by propidium iodide (PI) assay. Apoptosis was analyzed by Wright-Giemsa, Hoechst 33342 and Annexin-V/PI staining assays. The change in mitochondrial membrane potential (MMP) was examined by JC-1 staining and flow cytometry. Expression of apoptosis-related proteins was determined by reverse transcription-polymerase chain reaction and Western blotting. Short interfering RNA (siRNA) against <it>Bcl-2 </it>was used in CD34⁺KG1a and Kasumi-1 cells incubated with/without DNR.</p> <p>Results</p> <p>Curcumin inhibited proliferation and induced apoptosis and G1/S arrest in both DNR-insensitive KG1a, Kasumi-1 and DNR-sensitive U937 cells. Curcumin-induced apoptosis was associated with reduced expression of both Bcl-2 mRNA and protein, subsequent loss of MMP, and activation of caspase-3 followed by PARP degradation. Curcumin synergistically enhanced the cytotoxic effect of DNR in DNR-insensitive KG1a and Kasumi-1 cells, consistent with decreased Bcl-2 expression. Accordingly, siRNA against <it>Bcl-2 </it>increased the susceptibility of KG1a and Kasumi-1 cells to DNR-induced apoptosis. More importantly, curcumin suppressed Bcl-2 expression, selectively inhibited proliferation and synergistically enhanced the cytotoxicity of DNR in primary CD34⁺AML cells, while showing limited lethality in normal CD34⁺hematopoietic progenitors.</p> <p>Conclusion</p> <p>Curcumin down-regulates Bcl-2 and induces apoptosis in DNR-insensitive CD34⁺AML cell lines and primary CD34⁺AML cells.</p

TCP Transcription Factors in Moso Bamboo (Phyllostachys edulis): Genome-Wide Identification and Expression Analysis

TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTORS (T), members of a plant-specific gene family, play significant roles during plant growth and development, as well as in response to environmental stress. However, knowledge about this family in moso bamboo (Phyllostachys edulis) is limited. Therefore, in this study, the first genome-wide identification, classification, characterization, and expression pattern analysis of the TCP transcription factor family in moso bamboo was performed. Sixteen TCP members were identified from the moso bamboo genome using a BLASTP algorithm-based method and verified using the Pfam database. Based on a multiple-sequence alignment, the members were divided into two subfamilies, and members of the same family shared highly conserved motif structures. Subcellular localization and transactivation activity analyses of four selected genes revealed that they were nuclear localized and had self-activation activities. Additionally, the expression levels of several PeTCP members were significantly upregulated under abscisic acid, methyl jasmonate, and salicylic acid treatments, indicating that they play crucial plant hormone transduction roles in the processes of plant growth and development, as well as in responses to environmental stresses. Thus, the current study provides previously lacking information on the TCP family in moso bamboo and reveals the potential functions of this gene family in growth and development