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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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MOSAIC: a chemical-genetic interaction data repository and web resource for exploring chemical modes of action.

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MOSAIC: a chemical-genetic interaction data repository and web resource for exploring chemical modes of action.

Bioinformatics. 2017 Nov 30;:

Authors: Nelson J, Simpkins SW, Safizadeh H, Li SC, Piotrowski JS, Hirano H, Yashiroda Y, Osada H, Yoshida M, Boone C, Myers CL

Abstract
Summary: Chemical-genomic approaches that map interactions between small molecules and genetic perturbations offer a promising strategy for functional annotation of uncharacterized bioactive compounds. We recently developed a new high-throughput platform for mapping chemical-genetic (CG) interactions in yeast that can be scaled to screen large compound collections, and we applied this system to generate CG interaction profiles for more than 13,000 compounds. When integrated with the existing global yeast genetic interaction network, CG interaction profiles can enable mode-of-action prediction for previously uncharacterized compounds as well as discover unexpected secondary effects for known drugs. To facilitate future analysis of these valuable data, we developed a public database and web interface named MOSAIC. The website provides a convenient interface for querying compounds, bioprocesses (GO terms), and genes for CG information including direct CG interactions, bioprocesses, and gene-level target predictions. MOSAIC also provides access to chemical structure information of screened molecules, chemical-genomic profiles, and the ability to search for compounds sharing structural and functional similarity. This resource will be of interest to chemical biologists for discovering new small molecule probes with specific modes-of-action as well as computational biologists interested in analyzing CG interaction networks.
Availability: MOSAIC is available at http://mosaic.cs.umn.edu.
Contact: chadm@umn.edu, charlie.boone@utoronto.ca, yoshidam@riken.jp, or hisyo@riken.jp.
Supplementary information: Supplementary data are available at Bioinformatics online.

PMID: 29206899 [PubMed - as supplied by publisher]



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Effects of Erythropoietin Receptor Activity on Angiogenesis, Tubular Injury and Fibrosis in Acute Kidney Injury: A "U-Shaped" Relationship.

Effects of Erythropoietin Receptor Activity on Angiogenesis, Tubular Injury and Fibrosis in Acute Kidney Injury: A "U-Shaped" Relationship.

Am J Physiol Renal Physiol. 2017 Nov 29;:ajprenal.00306.2017

Authors: Shi M, Flores B, Li P, Gillings N, McMillan KL, Ye J, Huang LJ, Sidhu SS, Zhong YP, Grompe MT, Streeter PR, Moe OW, Hu MC

Abstract
Erythropoietin receptor (EpoR) is widely expressed but its renoprotective action is unexplored. To examine the role of EpoR in vivo in the kidney, we induced acute kidney injury (AKI) by ischemia-reperfusion in mice with different EpoR bioactivities in the kidney. EpoR bioactivity was reduced by knock-in of wild type human EpoR, which is hypo-functional relative to murine EpoR, and a renal tubule-specific EpoR knockout. These mice had lower EPO/EpoR activity and lower autophagy flux in renal tubules. Upon AKI induction, they exhibited worse renal function and structural damage, and more apoptosis at the acute stage (< 7 days), and slower recovery with more tubulointerstitial fibrosis at the subacute stage (14 days). In contrast, mice with hyperactive EpoR signaling from knock-in of a constitutively active human EpoR had higher autophagic flux, milder kidney damage and better renal function at the acute stage, but surprisingly, worse tubulointerstitial fibrosis and renal function at the subacute stage. Either excess or deficient EpoR activity in the kidney was associated with abnormal peritubular capillaries and tubular hypoxia, creating a "U-shape" relationship. The direct effects of EpoR on tubular cells were confirmed ex vivo by a hydrogen peroxide model using primary cultured proximal tubule cells with different EpoR activities. In summary, normal EPO/EpoR signaling in renal tubules provides defense against renal tubular injury, maintains the autophagy-apoptosis balance and peritubular capillary integrity. High and low EPO/EpoR bioactivities both lead to vascular defect, and high EpoR activity overides the tubular protective effects in AKI recovery.

PMID: 29187371 [PubMed - as supplied by publisher]



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CRISPR/Cas9 System as a Valuable Genome Editing Tool for Wine Yeasts with Application to Decrease Urea Production.

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CRISPR/Cas9 System as a Valuable Genome Editing Tool for Wine Yeasts with Application to Decrease Urea Production.

Front Microbiol. 2017;8:2194

Authors: Vigentini I, Gebbia M, Belotti A, Foschino R, Roth FP

Abstract
An extensive repertoire of molecular tools is available for genetic analysis in laboratory strains of S. cerevisiae. Although this has widely contributed to the interpretation of gene functionality within haploid laboratory isolates, the genetics of metabolism in commercially-relevant polyploid yeast strains is still poorly understood. Genetic engineering in industrial yeasts is undergoing major changes due to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) engineering approaches. Here we apply the CRISPR/Cas9 system to two commercial "starter" strains of S. cerevisiae (EC1118, AWRI796), eliminating the CAN1 arginine permease pathway to generate strains with reduced urea production (18.5 and 35.5% for EC1118 and AWRI796, respectively). In a wine-model environment based on two grape musts obtained from Chardonnay and Cabernet Sauvignon cultivars, both S. cerevisiae starter strains and CAN1 mutants completed the must fermentation in 8-12 days. However, recombinant strains carrying the can1 mutation failed to produce urea, suggesting that the genetic modification successfully impaired the arginine metabolism. In conclusion, the reduction of urea production in a wine-model environment confirms that the CRISPR/Cas9 system has been successfully established in S. cerevisiae wine yeasts.

PMID: 29163459 [PubMed]



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High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification.

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High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification.

J Vis Exp. 2017 Oct 19;(128):

Authors: Jandura A, Hu J, Wilk R, Krause HM

Abstract
In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

PMID: 29155736 [PubMed - in process]



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