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Recent Publications

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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Structural basis for arginine methylation-independent recognition of PIWIL1 by TDRD2.

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Structural basis for arginine methylation-independent recognition of PIWIL1 by TDRD2.

Proc Natl Acad Sci U S A. 2017 11 21;114(47):12483-12488

Authors: Zhang H, Liu K, Izumi N, Huang H, Ding D, Ni Z, Sidhu SS, Chen C, Tomari Y, Min J

Abstract
The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway plays a central role in transposon silencing and genome protection in the animal germline. A family of Tudor domain proteins regulates the piRNA pathway through direct Tudor domain-PIWI interactions. Tudor domains are known to fulfill this function by binding to methylated PIWI proteins in an arginine methylation-dependent manner. Here, we report a mechanism of methylation-independent Tudor domain-PIWI interaction. Unlike most other Tudor domains, the extended Tudor domain of mammalian Tudor domain-containing protein 2 (TDRD2) preferentially recognizes an unmethylated arginine-rich sequence from PIWI-like protein 1 (PIWIL1). Structural studies reveal an unexpected Tudor domain-binding mode for the PIWIL1 sequence in which the interface of Tudor and staphylococcal nuclease domains is primarily responsible for PIWIL1 peptide recognition. Mutations disrupting the TDRD2-PIWIL1 interaction compromise piRNA maturation via 3'-end trimming in vitro. Our work presented here reveals the molecular divergence of the interactions between different Tudor domain proteins and PIWI proteins.

PMID: 29118143 [PubMed - indexed for MEDLINE]



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Construction of Synthetic Antibody Phage-Display Libraries.

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Construction of Synthetic Antibody Phage-Display Libraries.

Methods Mol Biol. 2018;1701:45-60

Authors: Nilvebrant J, Sidhu SS

Abstract
Synthetic antibody libraries provide a vast resource of renewable antibody reagents that can rival or exceed those of natural antibodies and can be rapidly isolated through controlled in vitro selections. Use of highly optimized human frameworks enables the incorporation of defined diversity at positions that are most likely to contribute to antigen recognition. This protocol describes the construction of synthetic antibody libraries based on a single engineered human autonomous variable heavy domain scaffold with diversity in all three complementarity-determining regions. The resulting libraries can be used to generate recombinant domain antibodies for a wide range of protein antigens using phage display. Furthermore, analogous methods can be used to construct antibody libraries based on larger antibody fragments or second-generation libraries aimed to fine-tune antibody characteristics including affinity, specificity, and manufacturability. The procedures rely on standard reagents and equipment available in most molecular biology laboratories.

PMID: 29116499 [PubMed - indexed for MEDLINE]



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A computational approach for designing D-proteins with non-canonical amino acid optimised binding affinity.

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A computational approach for designing D-proteins with non-canonical amino acid optimised binding affinity.

PLoS One. 2017;12(11):e0187524

Authors: Garton M, Sayadi M, Kim PM

Abstract
Redesigning protein surface topology to improve target binding holds great promise in the search for highly selective therapeutics. While significant binding improvements can be achieved using natural amino acids, the introduction of non-canonical residues vastly increases sequence space and thus the chance to significantly out-compete native partners. The potency of protein inhibitors can be further enhanced by synthesising mirror image, D-amino versions. This renders them non-immunogenic and makes them highly resistant to proteolytic degradation. Current experimental design methods often preclude the use of D-amino acids and non-canonical amino acids for a variety of reasons. To address this, we build an in silico pipeline for D-protein designs featuring non-canonical amino acids. For a test scaffold we use an existing D-protein inhibitor of VEGF: D-RFX001. We benchmark the approach by recapitulating previous experimental optimisation with canonical amino acids. Subsequent incorporation of non-canonical amino acids allows designs that are predicted to improve binding affinity by up to -7.18 kcal/mol.

PMID: 29108013 [PubMed - indexed for MEDLINE]



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The influence of microRNAs and poly(A) tail length on endogenous mRNA-protein complexes.

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The influence of microRNAs and poly(A) tail length on endogenous mRNA-protein complexes.

Genome Biol. 2017 Oct 31;18(1):211

Authors: Rissland OS, Subtelny AO, Wang M, Lugowski A, Nicholson B, Laver JD, Sidhu SS, Smibert CA, Lipshitz HD, Bartel DP

Abstract
BACKGROUND: All mRNAs are bound in vivo by proteins to form mRNA-protein complexes (mRNPs), but changes in the composition of mRNPs during posttranscriptional regulation remain largely unexplored. Here, we have analyzed, on a transcriptome-wide scale, how microRNA-mediated repression modulates the associations of the core mRNP components eIF4E, eIF4G, and PABP and of the decay factor DDX6 in human cells.
RESULTS: Despite the transient nature of repressed intermediates, we detect significant changes in mRNP composition, marked by dissociation of eIF4G and PABP, and by recruitment of DDX6. Furthermore, although poly(A)-tail length has been considered critical in post-transcriptional regulation, differences in steady-state tail length explain little of the variation in either PABP association or mRNP organization more generally. Instead, relative occupancy of core components correlates best with gene expression.
CONCLUSIONS: These results indicate that posttranscriptional regulatory factors, such as microRNAs, influence the associations of PABP and other core factors, and do so without substantially affecting steady-state tail length.

PMID: 29089021 [PubMed - indexed for MEDLINE]



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