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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 - indexed for MEDLINE]



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SGK1 (glucose transport), dishevelled2 (wnt signaling), LC3/p62 (autophagy) and p53 (apoptosis) proteins are unaltered in Lafora disease.

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SGK1 (glucose transport), dishevelled2 (wnt signaling), LC3/p62 (autophagy) and p53 (apoptosis) proteins are unaltered in Lafora disease.

All Results J Biol. 2016;7(3):28-33

Authors: Wang P, Israelian L, Xue Y, Song S, Attisano L, Minassian BA

Abstract
Glycogen forms through the concerted actions of glycogen synthase (GS) which elongates glycogen strands, and glycogen branching enzyme (GBE). Lafora disease (LD) is a fatal neurodegenerative epilepsy that results from neuronal accumulation of hyperphosphorylated glycogen with excessively long strands (called polyglucosans). There is no GBE deficiency in LD. Instead, the disease is caused by loss-of-function mutations in the EPM2A or EPM2B genes, encoding, respectively, a phosphatase, laforin, and an E3 ubiquiting ligase, malin. A number of experimentally derived hypotheses have been published to explain LD, including: The SGK1 hypothesis - Phosphorylated SGK1 (pSGK1) raises cellular glucose uptake and levels, which would activate GS. Based on observing increased pSGK1 in LD mice it was proposed that raised pSGK1 leads to polyglucosan generation through GS hyperactivation. The Dishevelled2 hypothesis - Downregulating malin in cell culture was reported to increase levels of dishevelled2, which through the wnt/glycogen synthase kinase-3 pathway would likewise overactivate GS. The Autophagic defect hypothesis - Polyglucosans may be natural byproducts of normal glycogen metabolism. LD mice were reported to be autophagy-defective. LD would arise from failed autophagy leading to failed polyglucosan clearance. Finally, the p53 hypothesis - laforin and malin were reported to downregulate p53, their absence leading to increased p53, which would activate apoptosis, leading to the neurodegeneration of LD. In the present work we repeat key experiments that underlie these four hypotheses. We are unable to confirm increased pSGK1, dishevelled2, or p53 in LD mice, nor the reported autophagic defects. Our work does not support the above hypotheses in understanding this unique and severe form of epilepsy.

PMID: 29152446 [PubMed]



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Comparison of ChIP-seq Data and a Reference Motif Set for Human KRAB C2H2 Zinc Finger Proteins.

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Comparison of ChIP-seq Data and a Reference Motif Set for Human KRAB C2H2 Zinc Finger Proteins.

G3 (Bethesda). 2017 Nov 16;:

Authors: Barazandeh M, Lambert S, Albu M, Hughes TR

Abstract
KRAB C2H2 zinc finger proteins (KZNFs) are the largest and most diverse family of human transcription factors, likely due to diversifying selection driven by novel endogenous retroelements (EREs), but the vast majority lack binding motifs or functional data. Two recent studies analyzed a majority of the human KZNFs using either ChIP-seq (60 proteins) or ChIP-exo (221 proteins) in the same cell type (HEK293). The ChIP-exo paper did not describe binding motifs, however. Thirty-nine proteins are represented in both studies, enabling the systematic comparison of the data sets presented here. Typically, only a minority of peaks overlap, but the two studies nonetheless display significant similarity in ERE binding for 32/39, and yield highly similar DNA binding motifs for 23 and related motifs for 34 (MoSBAT similarity score > 0.5 and > 0.2, respectively). Thus, there is overall (albeit imperfect) agreement between the two studies. For the 242 proteins represented in at least one study, we selected a highest-confidence motif for each protein, utilizing several motif-derivation approaches, and evaluating motifs within and across data sets. Peaks for the majority (158) are enriched (96% with AUC > 0.6 predicting peak vs non-peak) for a motif that is supported by the C2H2 "recognition code", consistent with intrinsic sequence specificity driving DNA binding in cells. An additional 63 yield motifs enriched in peaks, but not supported by the recognition code, which could reflect indirect binding. Altogether, these analyses validate both data sets, and provide a reference motif set with associated quality metrics.

PMID: 29146583 [PubMed - as supplied by publisher]



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A Map of Human Mitochondrial Protein Interactions Linked to Neurodegeneration Reveals New Mechanisms of Redox Homeostasis and NF-κB Signaling.

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A Map of Human Mitochondrial Protein Interactions Linked to Neurodegeneration Reveals New Mechanisms of Redox Homeostasis and NF-κB Signaling.

Cell Syst. 2017 Nov 07;:

Authors: Malty RH, Aoki H, Kumar A, Phanse S, Amin S, Zhang Q, Minic Z, Goebels F, Musso G, Wu Z, Abou-Tok H, Meyer M, Deineko V, Kassir S, Sidhu V, Jessulat M, Scott NE, Xiong X, Vlasblom J, Prasad B, Foster LJ, Alberio T, Garavaglia B, Yu H, Bader GD, Nakamura K, Parkinson J, Babu M

Abstract
Mitochondrial protein (MP) dysfunction has been linked to neurodegenerative disorders (NDs); however, the discovery of the molecular mechanisms underlying NDs has been impeded by the limited characterization of interactions governing MP function. Here, using mass spectrometry (MS)-based analysis of 210 affinity-purified mitochondrial (mt) fractions isolated from 27 epitope-tagged human ND-linked MPs in HEK293 cells, we report a high-confidence MP network including 1,964 interactions among 772 proteins (>90% previously unreported). Nearly three-fourths of these interactions were confirmed in mouse brain and multiple human differentiated neuronal cell lines by primary antibody immunoprecipitation and MS, with many linked to NDs and autism. We show that the SOD1-PRDX5 interaction, critical for mt redox homeostasis, can be perturbed by amyotrophic lateral sclerosis-linked SOD1 allelic variants and establish a functional role for ND-linked factors coupled with IκBɛ in NF-κB activation. Our results identify mechanisms for ND-linked MPs and expand the human mt interaction landscape.

PMID: 29128334 [PubMed - as supplied by publisher]



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The combinatorial control of alternative splicing in C. elegans.

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The combinatorial control of alternative splicing in C. elegans.

PLoS Genet. 2017 Nov;13(11):e1007033

Authors: Tan JH, Fraser AG

Abstract
Normal development requires the right splice variants to be made in the right tissues at the right time. The core splicing machinery is engaged in all splicing events, but which precise splice variant is made requires the choice between alternative splice sites-for this to occur, a set of splicing factors (SFs) must recognize and bind to short RNA motifs in the pre-mRNA. In C. elegans, there is known to be extensive variation in splicing patterns across development, but little is known about the targets of each SF or how multiple SFs combine to regulate splicing. Here we combine RNA-seq with in vitro binding assays to study how 4 different C. elegans SFs, ASD-1, FOX-1, MEC-8, and EXC-7, regulate splicing. The 4 SFs chosen all have well-characterised biology and well-studied loss-of-function genetic alleles, and all contain RRM domains. Intriguingly, while the SFs we examined have varied roles in C. elegans development, they show an unexpectedly high overlap in their targets. We also find that binding sites for these SFs occur on the same pre-mRNAs more frequently than expected suggesting extensive combinatorial control of splicing. We confirm that regulation of splicing by multiple SFs is often combinatorial and show that this is functionally significant. We also find that SFs appear to combine to affect splicing in two modes-they either bind in close proximity within the same intron or they appear to bind to separate regions of the intron in a conserved order. Finally, we find that the genes whose splicing are regulated by multiple SFs are highly enriched for genes involved in the cytoskeleton and in ion channels that are key for neurotransmission. Together, this shows that specific classes of genes have complex combinatorial regulation of splicing and that this combinatorial regulation is critical for normal development to occur.

PMID: 29121637 [PubMed - indexed for MEDLINE]



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The Role of Nanoparticle Design in Determining Analytical Performance of Lateral Flow Immunoassays.

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The Role of Nanoparticle Design in Determining Analytical Performance of Lateral Flow Immunoassays.

Nano Lett. 2017 Nov 15;:

Authors: Zhan L, Guo SZ, Song F, Gong Y, Xu F, Boulware DR, McAlpine MC, Chan WCW, Bischof JC

Abstract
Rapid, simple, and cost-effective diagnostics are needed to improve healthcare at the point of care (POC). However, the most widely used POC diagnostic, the lateral flow immunoassay (LFA), is ∼1000-times less sensitive and has a smaller analytical range than laboratory tests, requiring a confirmatory test to establish truly negative results. Here, a rational and systematic strategy is used to design the LFA contrast label (i.e., gold nanoparticles) to improve the analytical sensitivity, analytical detection range, and antigen quantification of LFAs. Specifically, we discovered that the size (30, 60, or 100 nm) of the gold nanoparticles is a main contributor to the LFA analytical performance through both the degree of receptor interaction and the ultimate visual or thermal contrast signals. Using the optimal LFA design, we demonstrated the ability to improve the analytical sensitivity by 256-fold and expand the analytical detection range from 3 log10 to 6 log10 for diagnosing patients with inflammatory conditions by measuring C-reactive protein. This work demonstrates that, with appropriate design of the contrast label, a simple and commonly used diagnostic technology can compete with more expensive state-of-the-art laboratory tests.

PMID: 29120648 [PubMed - as supplied by publisher]



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