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DNA Sequence Recognition of Human CXXC Domains and Their Structural Determinants.

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DNA Sequence Recognition of Human CXXC Domains and Their Structural Determinants.

Structure. 2017 Dec 16;:

Authors: Xu C, Liu K, Lei M, Yang A, Li Y, Hughes TR, Min J

Abstract
The CXXC domain, first identified as the reader of unmodified CpG dinucleotide, plays important roles in epigenetic regulation by targeting various activities to CpG islands. Here we systematically measured and compared the DNA-binding selectivities of all known human CXXC domains by different binding assays, and complemented the existing function-based classification of human CXXC domains with a classification based on their DNA selectivities. Through a series of crystal structures of CXXC domains with DNA ligands, we unravel the molecular mechanisms of how these CXXC domains, including single CXXC domains and tandem CXXC-PHD domains, recognize distinct DNA ligands, which further supports our classification of human CXXC domains and also provides insights into selective recruitment of chromatin modifiers to their respective targets via CXXC domains recognizing different genomic DNA sequences. Our study facilitates the understanding of the relationship between the DNA-binding specificities of the CXXC proteins and their biological functions.

PMID: 29276034 [PubMed - as supplied by publisher]



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A framework for exhaustively mapping functional missense variants.

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A framework for exhaustively mapping functional missense variants.

Mol Syst Biol. 2017 Dec 21;13(12):957

Authors: Weile J, Sun S, Cote AG, Knapp J, Verby M, Mellor JC, Wu Y, Pons C, Wong C, van Lieshout N, Yang F, Tasan M, Tan G, Yang S, Fowler DM, Nussbaum R, Bloom JD, Vidal M, Hill DE, Aloy P, Roth FP

Abstract
Although we now routinely sequence human genomes, we can confidently identify only a fraction of the sequence variants that have a functional impact. Here, we developed a deep mutational scanning framework that produces exhaustive maps for human missense variants by combining random codon mutagenesis and multiplexed functional variation assays with computational imputation and refinement. We applied this framework to four proteins corresponding to six human genes: UBE2I (encoding SUMO E2 conjugase), SUMO1 (small ubiquitin-like modifier), TPK1 (thiamin pyrophosphokinase), and CALM1/2/3 (three genes encoding the protein calmodulin). The resulting maps recapitulate known protein features and confidently identify pathogenic variation. Assays potentially amenable to deep mutational scanning are already available for 57% of human disease genes, suggesting that DMS could ultimately map functional variation for all human disease genes.

PMID: 29269382 [PubMed - in process]



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Enhanced human hematopoietic stem and progenitor cell engraftment by blocking donor T cell-mediated TNFα signaling.

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Enhanced human hematopoietic stem and progenitor cell engraftment by blocking donor T cell-mediated TNFα signaling.

Sci Transl Med. 2017 Dec 20;9(421):

Authors: Wang W, Fujii H, Kim HJ, Hermans K, Usenko T, Xie S, Luo ZJ, Ma J, Celso CL, Dick JE, Schroeder T, Krueger J, Wall D, Egeler RM, Zandstra PW

Abstract
Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy, but the large number of HSCs required limits its widespread use. Host conditioning and donor cell composition are known to affect HSCT outcomes. However, the specific role that the posttransplantation signaling environment plays in donor HSC fate is poorly understood. To mimic clinical HSCT, we injected human umbilical cord blood (UCB) cells at different doses and compositions into immunodeficient NOD/SCID/IL-2Rgc-null (NSG) mice. Surprisingly, higher UCB cell doses inversely correlated with stem and progenitor cell engraftment. This observation was attributable to increased donor cell-derived inflammatory signals. Donor T cell-derived tumor necrosis factor-α (TNFα) was specifically found to directly impair the survival and division of transplanted HSCs and progenitor cells. Neutralizing donor T cell-derived TNFα in vivo increased short-term stem and progenitor cell engraftment, accelerated hematopoietic recovery, and altered donor immune cell compositions. This direct effect of TNFα on transplanted cells could be decoupled from the indirect effect of alleviating graft-versus-host disease (GVHD) by interleukin-6 (IL-6) blockade. Our study demonstrates that donor immune cell-derived inflammatory signals directly influence HSC fate, and provides new clinically relevant strategies to improve engraftment efficiency during HSCT.

PMID: 29263228 [PubMed - in process]



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FZD4 Marks Lateral Plate Mesoderm and Signals with NORRIN to Increase Cardiomyocyte Induction from Pluripotent Stem Cell-Derived Cardiac Progenitors.

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FZD4 Marks Lateral Plate Mesoderm and Signals with NORRIN to Increase Cardiomyocyte Induction from Pluripotent Stem Cell-Derived Cardiac Progenitors.

Stem Cell Reports. 2017 Dec 13;:

Authors: Yoon C, Song H, Yin T, Bausch-Fluck D, Frei AP, Kattman S, Dubois N, Witty AD, Hewel JA, Guo H, Emili A, Wollscheid B, Keller G, Zandstra PW

Abstract
The identification of cell surface proteins on stem cells or stem cell derivatives is a key strategy for the functional characterization, isolation, and understanding of stem cell population dynamics. Here, using an integrated mass spectrometry- and microarray-based approach, we analyzed the surface proteome and transcriptome of cardiac progenitor cells (CPCs) generated from the stage-specific differentiation of mouse and human pluripotent stem cells. Through bioinformatics analysis, we have identified and characterized FZD4 as a marker for lateral plate mesoderm. Additionally, we utilized FZD4, in conjunction with FLK1 and PDGFRA, to further purify CPCs and increase cardiomyocyte (CM) enrichment in both mouse and human systems. Moreover, we have shown that NORRIN presented to FZD4 further increases CM output via proliferation through the canonical WNT pathway. Taken together, these findings demonstrate a role for FZD4 in mammalian cardiac development.

PMID: 29249665 [PubMed - as supplied by publisher]



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Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein.

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Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein.

MBio. 2017 Dec 12;8(6):

Authors: Pawluk A, Shah M, Mejdani M, Calmettes C, Moraes TF, Davidson AR, Maxwell KL

Abstract
CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor.IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system.

PMID: 29233895 [PubMed - in process]



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The nitrogen regulatory PII protein (GlnB) and N-acetyl-glucosamine 6-phosphate epimerase (NanE) allosterically activate glucosamine 6-phosphate deaminase (NagB) in Escherichia coli.

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The nitrogen regulatory PII protein (GlnB) and N-acetyl-glucosamine 6-phosphate epimerase (NanE) allosterically activate glucosamine 6-phosphate deaminase (NagB) in Escherichia coli.

J Bacteriol. 2017 Dec 11;:

Authors: Rodionova IA, Goodacre N, Babu M, Emili A, Uetz P, Saier MH

Abstract
Amino sugars are good sources of both ammonia and fructose-6-phosphate, produced by glucosamine 6-phosphate deaminase, NagB. NagB is known to be allosterically regulated by N-acetyl-glucosamine 6-phosphate (GlcNAc-6P) and the phosphocarrier protein of the bacterial phosphotransferase system, HPr, in Escherichia coli We provide evidence that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein also allosterically activate NagB by direct protein-protein interactions. NanE is essential for neuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and PII is known to be a central metabolic nitrogen regulator. We demonstrate that uridylylated PII (but not underivatized PII) activates NagB more then 10-fold at low concentrations of substrate, while NanE increases NagB activity over 2 fold. NanE activates NagB in the absence or presence of GlcNAc-6P, but HPr and U-PII activation requires the presence of GlcNAc-6P. Activation of NagB by HPr and uridylylated PII as well as by NanE and HPr (but not by NanE and U-PII) is synergistic, and the modelling, which suggests specific residues involved in complex formation, provides possible explanations. Specific physiological functions for the regulation of NagB by its three protein activators are proposed. Each regulatory agent is suggested to mediate signal transduction in response to a different stimulus.IMPORTANCE The regulation of amino sugar utilization is important for the survival of bacteria in a competitive environment. NagB, glucosamine 6-phosphate deaminase in E. coli, is essential for amino sugar utilization and is known to be allosterically regulated by N-acetyl-glucosamine 6-phosphate (GlcNAc-6P) and the histidine-phosphorylatable phosphocarrier protein, HPr. We provide evidence that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein allosterically activate NagB by direct protein-protein interactions. NanE is essential for N-acetylneuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and the PII protein is known to be a central metabolic nitrogen regulator. Regulatory links between carbon and nitrogen metabolism are important for adaptation of metabolism to different growth conditions.

PMID: 29229699 [PubMed - as supplied by publisher]



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The Role of Sulfur Dioxide in Stratospheric Aerosol Formation Evaluated Using In-Situ Measurements in the Tropical Lower Stratosphere.

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The Role of Sulfur Dioxide in Stratospheric Aerosol Formation Evaluated Using In-Situ Measurements in the Tropical Lower Stratosphere.

Geophys Res Lett. 2017 May 16;44(9):4280-4286

Authors: Rollins AW, Thornberry TD, Watts LA, Yu P, Rosenlof KH, Mills M, Baumann E, Giorgetta FR, Bui TV, Höpfner M, Walker KA, Boone C, Bernath PF, Colarco PR, Newman PA, Fahey DW, Gao RS

Abstract
Stratospheric aerosols (SAs) are a variable component of the Earth's albedo that may be intentionally enhanced in the future to offset greenhouse gases (geoengineering). The role of tropospheric-sourced sulfur dioxide (SO2) in maintaining background SAs has been debated for decades without in-situ measurements of SO2 at the tropical tropopause to inform this issue. Here we clarify the role of SO2 in maintaining SAs by using new in-situ SO2 measurements to evaluate climate models and satellite retrievals. We then use the observed tropical tropopause SO2 mixing ratios to estimate the global flux of SO2 across the tropical tropopause. These analyses show that the tropopause background SO2 is about 5 times smaller than reported by the average satellite observations that have been used recently to test atmospheric models. This shifts the view of SO2 as a dominant source of SAs to a near-negligible one, possibly revealing a significant gap in the SA budget.

PMID: 29225384 [PubMed - in process]



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The intracellular immune receptor Rx1 regulates the DNA-binding activity of a Golden2-like transcription factor.

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The intracellular immune receptor Rx1 regulates the DNA-binding activity of a Golden2-like transcription factor.

J Biol Chem. 2017 Dec 07;:

Authors: Townsend PD, Dixon CH, Slootweg EJ, Sukarta OC, Yang AW, Hughes TR, Sharples GJ, Palsson LO, Takken FLW, Goverse A, Cann MJ

Abstract
Plant NLR proteins enable the immune system to recognise and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming and some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato is further able to bind and distort double-stranded DNA. However, Rx1 host targets that support a role for Rx1 in transcriptional reprogramming at DNA are unknown. Here we report a functional interaction between Rx1 and NbGlk1, a Golden2- like transcription factor. Rx1 binds to NbGlk1 in vitro and in planta. NbGlk1 binds to known Golden2-like consensus DNA sequences. Rx1 reduces the binding affinity of NbGlk1 for DNA in vitro. NbGlk1 activates cellular responses to potato virus X, whereas Rx1 associates with NbGlk1 and prevents its assembly on DNA in planta unless activated by PVX. This study provides new mechanistic insight into how an NLR can co-ordinate an immune signalling response at DNA following pathogen perception.

PMID: 29217772 [PubMed - as supplied by publisher]



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Panomics for Precision Medicine.

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Panomics for Precision Medicine.

Trends Mol Med. 2017 Dec 04;:

Authors: Sandhu C, Qureshi A, Emili A

Abstract
Medicine is poised to undergo a digital transformation. High-throughput platforms are creating terabytes of genomic, transcriptomic, proteomic, and metabolomic data. The challenge is to interpret these data in a meaningful manner - to uncover relationships that are not readily apparent between molecular profiles and states of health or disease. This will require the development of novel data pipelines and computational tools. The combined analysis of multi-dimensional data is referred to as 'panomics'. The ultimate hope of integrative panomics is that it will lead to the discovery and application of novel markers and targeted therapeutics that drive forward a new era of 'precision medicine' where inter-individual variation is accounted for in the treatment of patients.

PMID: 29217119 [PubMed - as supplied by publisher]



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Effect of removing Kupffer cells on nanoparticle tumor delivery.

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Effect of removing Kupffer cells on nanoparticle tumor delivery.

Proc Natl Acad Sci U S A. 2017 Dec 05;:

Authors: Tavares AJ, Poon W, Zhang YN, Dai Q, Besla R, Ding D, Ouyang B, Li A, Chen J, Zheng G, Robbins C, Chan WCW

Abstract
A recent metaanalysis shows that 0.7% of nanoparticles are delivered to solid tumors. This low delivery efficiency has major implications in the translation of cancer nanomedicines, as most of the nanomedicines are sequestered by nontumor cells. To improve the delivery efficiency, there is a need to investigate the quantitative contribution of each organ in blocking the transport of nanoparticles to solid tumors. Here, we hypothesize that the removal of the liver macrophages, cells that have been reported to take up the largest amount of circulating nanoparticles, would lead to a significant increase in the nanoparticle delivery efficiency to solid tumors. We were surprised to discover that the maximum achievable delivery efficiency was only 2%. In our analysis, there was a clear correlation between particle design, chemical composition, macrophage depletion, tumor pathophysiology, and tumor delivery efficiency. In many cases, we observed an 18-150 times greater delivery efficiency, but we were not able to achieve a delivery efficiency higher than 2%. The results suggest the need to look deeper at other organs such as the spleen, lymph nodes, and tumor in mediating the delivery process. Systematically mapping the contribution of each organ quantitatively will allow us to pinpoint the cause of the low tumor delivery efficiency. This, in effect, enables the generation of a rational strategy to improve the delivery efficiency of nanoparticles to solid tumors either through the engineering of multifunctional nanosystems or through manipulation of biological barriers.

PMID: 29208719 [PubMed - as supplied by publisher]



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