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Controlling DNA-nanoparticle serum interactions.

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Controlling DNA-nanoparticle serum interactions.

Proc Natl Acad Sci U S A. 2016 Nov 16;:

Authors: Zagorovsky K, Chou LY, Chan WC

Abstract
Understanding the interaction of molecularly assembled nanoparticles with physiological fluids is critical to their use for in vivo delivery of drugs and contrast agents. Here, we systematically investigated the factors and mechanisms that govern the degradation of DNA on the nanoparticle surface in serum. We discovered that a higher DNA density, shorter oligonucleotides, and thicker PEG layer increased protection of DNA against serum degradation. Oligonucleotides on the surface of nanoparticles were highly resistant to DNase I endonucleases, and degradation was carried out exclusively by protein-mediated exonuclease cleavage and full-strand desorption. These results enabled the programming of the degradation rates of the DNA-assembled nanoparticle system from 0.1 to 0.7 h(-1) and the engineering of superstructures that can release two different preloaded dye molecules with distinct kinetics and half-lives ranging from 3.3 to 9.8 h. This study provides a general framework for investigating the serum stability of DNA-containing nanostructures. The results advance our understanding of engineering principles for designing nanoparticle assemblies with controlled in vivo behavior and present a strategy for storage and multistage release of drugs and contrast agents that can facilitate the diagnosis and treatment of cancer and other diseases.

PMID: 27856755 [PubMed - as supplied by publisher]



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Transcription Factors Encoded on Core and Accessory Chromosomes of Fusarium oxysporum Induce Expression of Effector Genes.

Transcription Factors Encoded on Core and Accessory Chromosomes of Fusarium oxysporum Induce Expression of Effector Genes.

PLoS Genet. 2016 Nov;12(11):e1006401

Authors: van der Does HC, Fokkens L, Yang A, Schmidt SM, Langereis L, Lukasiewicz JM, Hughes TR, Rep M

Abstract
Proteins secreted by pathogens during host colonization largely determine the outcome of pathogen-host interactions and are commonly called 'effectors'. In fungal plant pathogens, coordinated transcriptional up-regulation of effector genes is a key feature of pathogenesis and effectors are often encoded in genomic regions with distinct repeat content, histone code and rate of evolution. In the tomato pathogen Fusarium oxysporum f. sp. lycopersici (Fol), effector genes reside on one of four accessory chromosomes, known as the 'pathogenicity' chromosome, which can be exchanged between strains through horizontal transfer. The three other accessory chromosomes in the Fol reference strain may also be important for virulence towards tomato. Expression of effector genes in Fol is highly up-regulated upon infection and requires Sge1, a transcription factor encoded on the core genome. Interestingly, the pathogenicity chromosome itself contains 13 predicted transcription factor genes and for all except one, there is a homolog on the core genome. We determined DNA binding specificity for nine transcription factors using oligonucleotide arrays. The binding sites for homologous transcription factors were highly similar, suggesting that extensive neofunctionalization of DNA binding specificity has not occurred. Several DNA binding sites are enriched on accessory chromosomes, and expression of FTF1, its core homolog FTF2 and SGE1 from a constitutive promoter can induce expression of effector genes. The DNA binding sites of only these three transcription factors are enriched among genes up-regulated during infection. We further show that Ftf1, Ftf2 and Sge1 can activate transcription from their binding sites in yeast. RNAseq analysis revealed that in strains with constitutive expression of FTF1, FTF2 or SGE1, expression of a similar set of plant-responsive genes on the pathogenicity chromosome is induced, including most effector genes. We conclude that the Fol pathogenicity chromosome may be partially transcriptionally autonomous, but there are also extensive transcriptional connections between core and accessory chromosomes.

PMID: 27855160 [PubMed - in process]



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Kin17 facilitates multiple double-strand break repair pathways that govern B cell class switching.

Kin17 facilitates multiple double-strand break repair pathways that govern B cell class switching.

Sci Rep. 2016 Nov 17;6:37215

Authors: Le MX, Haddad D, Ling AK, Li C, So CC, Chopra A, Hu R, Angulo JF, Moffat J, Martin A

Abstract
Class switch recombination (CSR) in B cells requires the timely repair of DNA double-stranded breaks (DSBs) that result from lesions produced by activation-induced cytidine deaminase (AID). Through a genome-wide RNAi screen, we identified Kin17 as a gene potentially involved in the maintenance of CSR in murine B cells. In this study, we confirm a critical role for Kin17 in CSR independent of AID activity. Furthermore, we make evident that DSBs generated by AID or ionizing radiation require Kin17 for efficient repair and resolution. Our report shows that reduced Kin17 results in an elevated deletion frequency following AID mutational activity in the switch region. In addition, deficiency in Kin17 affects the functionality of multiple DSB repair pathways, namely homologous recombination, non-homologous end-joining, and alternative end-joining. This report demonstrates the importance of Kin17 as a critical factor that acts prior to the repair phase of DSB repair and is of bona fide importance for CSR.

PMID: 27853268 [PubMed - in process]



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Multiparameter functional diversity of human C2H2 zinc finger proteins.

Multiparameter functional diversity of human C2H2 zinc finger proteins.

Genome Res. 2016 Nov 16;

Authors: Schmitges FW, Radovani E, Najafabadi HS, Barazandeh M, Campitelli LF, Yin Y, Jolma A, Zhong G, Guo H, Kanagalingam T, Dai WF, Taipale J, Emili A, Greenblatt JF, Hughes TR

Abstract
C2H2 zinc finger proteins represent the largest and most enigmatic class of human transcription factors. Their C2H2-ZF arrays are highly variable, indicating that most will have unique DNA binding motifs. However, most of the binding motifs have not been directly determined. In addition, little is known about whether or how these proteins regulate transcription. Most of the ∼700 human C2H2-ZF proteins also contain at least one KRAB, SCAN, BTB, or SET domain, suggesting that they may have common interacting partners and/or effector functions. Here, we report a multifaceted functional analysis of 131 human C2H2-ZF proteins, encompassing DNA binding sites, interacting proteins, and transcriptional response to genetic perturbation. We confirm the expected diversity in DNA binding motifs and genomic binding sites, and provide motif models for 78 previously uncharacterized C2H2-ZF proteins, most of which are unique. Surprisingly, the diversity in protein-protein interactions is nearly as high as diversity in DNA binding motifs: Most C2H2-ZF proteins interact with a unique spectrum of co-activators and co-repressors. Thus, multiparameter diversification likely underlies the evolutionary success of this large class of human proteins.

PMID: 27852650 [PubMed - as supplied by publisher]



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Pan-neuronal screening in Caenorhabditis elegans reveals asymmetric dynamics of AWC neurons is critical for thermal avoidance behavior.

Pan-neuronal screening in Caenorhabditis elegans reveals asymmetric dynamics of AWC neurons is critical for thermal avoidance behavior.

Elife. 2016 Nov 16;5:

Authors: Kotera I, Tran NA, Fu D, Kim JH, Byrne Rodgers JD, Ryu WS

Abstract
Understanding neural functions inevitably involves arguments traversing multiple levels of hierarchy in biological systems. However, finding new components or mechanisms of such systems is extremely time-consuming due to the low efficiency of currently available functional screening techniques. To overcome such obstacles, we utilize pan-neuronal calcium imaging to broadly screen the activity of the C. elegans nervous system in response to thermal stimuli. A single pass of the screening procedure can identify much of the previously reported thermosensory circuitry as well as identify several unreported thermosensory neurons. Among the newly discovered neural functions, we investigated the role of the AWC(OFF) neuron in thermal nociception. Combining functional calcium imaging and behavioral assays, we show that AWC(OFF) is essential for avoidance behavior following noxious heat stimulation by modifying the forward-to-reversal behavioral transition rate. We also show that the AWC(OFF) signals adapt to repeated noxious thermal stimuli and quantify the corresponding behavioral adaptation.

PMID: 27849153 [PubMed - as supplied by publisher]



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Ten Simple Rules for Developing Public Biological Databases.

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Ten Simple Rules for Developing Public Biological Databases.

PLoS Comput Biol. 2016 Nov;12(11):e1005128

Authors: Helmy M, Crits-Christoph A, Bader GD

PMID: 27832061 [PubMed - in process]



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AutoAnnotate: A Cytoscape app for summarizing networks with semantic annotations.

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AutoAnnotate: A Cytoscape app for summarizing networks with semantic annotations.

F1000Res. 2016;5:1717

Authors: Kucera M, Isserlin R, Arkhangorodsky A, Bader GD

Abstract
Networks often contain regions of tightly connected nodes, or clusters, that highlight their shared relationships. An effective way to create a visual summary of a network is to identify clusters and annotate them with an enclosing shape and a summarizing label. Cytoscape provides the ability to annotate a network with shapes and labels, however these annotations must be created manually one at a time, which can be a laborious process. AutoAnnotate is a Cytoscape 3 App that automates the process of identifying clusters and visually annotating them. It greatly reduces the time and effort required to fully annotate clusters in a network, and provides freedom to experiment with different strategies for identifying and labelling clusters. Many customization options are available that enable the user to refine the generated annotations as required. Annotated clusters may be collapsed into single nodes using the Cytoscape groups feature, which helps simplify a network by making its overall structure more visible. AutoAnnotate is applicable to any type of network, including enrichment maps, protein-protein interactions, pathways, or social networks.

PMID: 27830058 [PubMed - in process]



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Distinct signaling programs control human hematopoietic stem cell survival and proliferation.

Distinct signaling programs control human hematopoietic stem cell survival and proliferation.

Blood. 2016 Nov 8;:

Authors: Knapp DJ, Hammond CA, Aghaeepour N, Miller PH, Pellacani D, Beer PA, Sachs K, Qiao W, Wang W, Humphries RK, Sauvageau G, Zandstra PW, Bendall SC, Nolan GP, Hansen C, Eaves CJ

Abstract
Several growth factors (GFs) that together promote quiescent human hematopoietic stem cell (HSC) expansion ex vivo have been identified. However, the molecular mechanisms by which these GFs regulate the survival, proliferation and differentiation of human HSCs remain poorly understood. We now describe experiments in which we used mass cytometry to simultaneously measure multiple surface markers, transcription factors, active signaling intermediates, viability and cell cycle indicators in single CD34(+) cord blood cells before and up to 2 hours after their stimulation with SCF, FLT3L, IL3, IL6, and G-CSF either alone or combined. Cells with a CD34(+)CD38(-)CD45RA(-)CD90(+)CD49f(+) (CD49f(+)) phenotype (~10% HSCs with >6 month repopulating activity in immunodeficient mice) displayed rapid increases in activated STAT1/3/5, ERK1/2, AKT, CREB, and S6 by one or more of these GFs, and β-catenin only when the 5 GFs were combined. Certain minority subsets within the CD49f(+) compartment were poorly GF-responsive and, among the more GF-responsive subsets of CD49f(+) cells, different signaling intermediates correlated with the levels of the myeloid- and lymphoid-associated transcription factors measured. Phenotypically similar, but CD90(-)CD49f(-) cells (MPPs) contained lower baseline levels of multiple signaling intermediates than the CD90(+)CD49f(+) cells, but showed similar response amplitudes to the same GFs. Importantly, we found activation or inhibition of AKT and β-catenin directly altered immediate CD49f(+) cell survival and proliferation. These findings identify rapid signaling events that 5 GFs elicit directly in the most primitive human hematopoietic cell types to promote their survival and proliferation.

PMID: 27827829 [PubMed - as supplied by publisher]



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Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication.

Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication.

PLoS Genet. 2016 Nov;12(11):e1006414

Authors: Shyian M, Mattarocci S, Albert B, Hafner L, Lezaja A, Costanzo M, Boone C, Shore D

Abstract
The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the rif1Δ sir2Δ double mutant shows no further increase in rDNA replication. Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2Δ. We find, in addition, that the viability of rif1Δ cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB) protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1Δ cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common.

PMID: 27820830 [PubMed - in process]



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The ribosome-engaged landscape of alternative splicing.

The ribosome-engaged landscape of alternative splicing.

Nat Struct Mol Biol. 2016 Nov 07;:

Authors: Weatheritt RJ, Sterne-Weiler T, Blencowe BJ

Abstract
High-throughput RNA sequencing (RNA-seq) has revealed an enormous complexity of alternative splicing (AS) across diverse cell and tissue types. However, it is currently unknown to what extent repertoires of splice-variant transcripts are translated into protein products. Here, we surveyed AS events engaged by the ribosome. Notably, at least 75% of human exon-skipping events detected in transcripts with medium-to-high abundance in RNA-seq data were also detected in ribosome profiling data. Furthermore, relatively small subsets of functionally related splice variants are engaged by ribosomes at levels that do not reflect their absolute abundance, thus indicating a role for AS in modulating translational output. This mode of regulation is associated with control of the mammalian cell cycle. Our results thus suggest that a major fraction of splice variants is translated and that specific cellular functions including cell-cycle control are subject to AS-dependent modulation of translation output.

PMID: 27820807 [PubMed - as supplied by publisher]



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