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A synthetic anti-Frizzled antibody engineered for broadened specificity exhibits enhanced anti-tumor properties.

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A synthetic anti-Frizzled antibody engineered for broadened specificity exhibits enhanced anti-tumor properties.

MAbs. 2018 Sep 05;:

Authors: Pavlovic Z, Adams JJ, Blazer LL, Gakhal AK, Jarvik N, Steinhart Z, Robitaille M, Mascall K, Pan J, Angers S, Moffat J, Sidhu SS

Abstract
Secreted Wnt ligands play a major role in the development and progression of many cancers by modulating signaling through cell-surface Frizzled receptors (FZDs). In order to achieve maximal effect on Wnt signaling by targeting the cell surface, we developed a synthetic antibody targeting six of the 10 human FZDs. We first identified an anti-FZD antagonist antibody (F2) with a specificity profile matching that of OMP-18R5, a monoclonal antibody that inhibits growth of many cancers by targeting FZD7, FZD1, FZD2, FZD5 and FZD8. We then used combinatorial antibody engineering by phage display to develop a variant antibody F2.A with specificity broadened to include FZD4. We confirmed that F2.A blocked binding of Wnt ligands, but not binding of Norrin, a ligand that also activates FZD4. Importantly, F2.A proved to be much more efficacious than either OMP-18R5 or F2 in inhibiting the growth of multiple RNF43-mutant pancreatic ductal adenocarcinoma cell lines, including patient-derived cells.

PMID: 30183492 [PubMed - as supplied by publisher]



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Single-Molecule Localization Microscopy Of Septin Bundles in Mammalian Cells.

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Single-Molecule Localization Microscopy Of Septin Bundles in Mammalian Cells.

Cytoskeleton (Hoboken). 2018 Sep 03;:

Authors: Vissa A, Giuliani M, Froese CD, Kim MS, Soroor F, Kim PK, Trimble WS, Yip CM

Abstract
Septins are a conserved family of GTPases that associate with numerous components of the cytoskeleton and the inner leaflet of the plasma membrane. These proteins are involved in many biological processes including cell division and membrane trafficking, and serving as a scaffolding component of the cytoskeleton used to recruit other proteins and form diffusion barriers to maintain the composition of membrane domains. In order to carry out their cellular functions, septins undergo interactions via their NC or G interfaces to form heteromeric rod-like structures that can polymerize into filaments and associate laterally into bundles. While electron microscopy studies of affinity-tagged and purified S. cerevisiae septin complexes have provided evidence for this periodic organization and in-registry lateral bundling in vitro, the in-vivo arrangement of stress fiber-associated septin bundles in mammalian cells remains poorly characterized. We report here on a Direct Stochastic Optical Reconstruction Microscopy (dSTORM) and Photoactivated Localization Microscopy (PALM) study of the 2D spatial distribution of septins in mammalian cells. From simulated and experimental results, we show the effects of labeling method, labeling efficiency, and fluorescent emitter photophysics on image reconstruction and interpretation. Our experimental results are consistent with septin organization by polymerization of hetero-octamers and an approximate 30-35 nm periodicity between subsequent units of SEPT2-SEPT2 or SEPT9-SEPT9. This article is protected by copyright. All rights reserved.

PMID: 30176126 [PubMed - as supplied by publisher]



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Motif models for RNA-binding proteins.

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Motif models for RNA-binding proteins.

Curr Opin Struct Biol. 2018 Aug 29;53:115-123

Authors: Sasse A, Laverty KU, Hughes TR, Morris QD

Abstract
Identifying the binding preferences of RNA-binding proteins (RBPs) is important in understanding their contribution to post-transcriptional regulation. Here, we review the current state-of-the art of RNA motif identification tools for RBPs. New in vivo and in vitro data sets provide sufficient statistical power to enable detection of relatively long and complex sequence and sequence-structure binding preferences, and recent computational methods are geared towards quantitative identification of these patterns. We classify methods by their motif model's representational power and describe the underlying considerations for RNA-protein interactions. All classical motif identification algorithms apply physically motivated architectures, consisting of a motif and an occupancy model, we call these explicit motif models. Recent methods, such as convolutional neural networks and support vector machines, abandon the classical architecture and implicitly model RNA binding without defining a motif model. Although they achieve high accuracy on held-out data they may be unsuitable to solve the ultimate goal of the field, using motifs trained on in vitro data to predict in vivo binding sites. For this task methods need to separate intrinsic binding preferences from cellular effects from protein and RNA concentrations, cooperativity, and competition. To tackle this problem, we advocate for the use of a `three-layer' architecture, consisting of motif model, occupancy model, and extrinsic factor model, which enables separation and adjustment to cellular conditions.

PMID: 30172081 [PubMed - as supplied by publisher]



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Idebenone and coenzyme Q10 are novel PPARα/γ ligands, with potential for treatment of fatty liver diseases.

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Idebenone and coenzyme Q10 are novel PPARα/γ ligands, with potential for treatment of fatty liver diseases.

Dis Model Mech. 2018 Aug 31;11(9):

Authors: Tiefenbach J, Magomedova L, Liu J, Reunov AA, Tsai R, Eappen NS, Jockusch RA, Nislow C, Cummins CL, Krause HM

Abstract
Current peroxisome proliferator-activated receptor (PPAR)-targeted drugs, such as the PPARγ-directed diabetes drug rosiglitazone, are associated with undesirable side effects due to robust agonist activity in non-target tissues. To find new PPAR ligands with fewer toxic effects, we generated transgenic zebrafish that can be screened in high throughput for new tissue-selective PPAR partial agonists. A structural analog of coenzyme Q10 (idebenone) that elicits spatially restricted partial agonist activity for both PPARα and PPARγ was identified. Coenzyme Q10 was also found to bind and activate both PPARs in a similar fashion, suggesting an endogenous role in relaying the states of mitochondria, peroxisomes and cellular redox to the two receptors. Testing idebenone in a mouse model of type 2 diabetes revealed the ability to reverse fatty liver development. These findings indicate new mechanisms of action for both PPARα and PPARγ, and new potential treatment options for nonalcoholic fatty liver disease (NAFLD) and steatosis.This article has an associated First Person interview with the first author of the paper.

PMID: 30171034 [PubMed - in process]



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A feed forward loop enforces YAP/TAZ signaling during tumorigenesis.

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A feed forward loop enforces YAP/TAZ signaling during tumorigenesis.

Nat Commun. 2018 08 29;9(1):3510

Authors: Gill MK, Christova T, Zhang YY, Gregorieff A, Zhang L, Narimatsu M, Song S, Xiong S, Couzens AL, Tong J, Krieger JR, Moran MF, Zlotta AR, van der Kwast TH, Gingras AC, Sicheri F, Wrana JL, Attisano L

Abstract
In most solid tumors, the Hippo pathway is inactivated through poorly understood mechanisms that result in the activation of the transcriptional regulators, YAP and TAZ. Here, we identify NUAK2 as a YAP/TAZ activator that directly inhibits LATS-mediated phosphorylation of YAP/TAZ and show that NUAK2 induction by YAP/TAZ and AP-1 is required for robust YAP/TAZ signaling. Pharmacological inhibition or loss of NUAK2 reduces the growth of cultured cancer cells and mammary tumors in mice. Moreover, in human patient samples, we show that NUAK2 expression is elevated in aggressive, high-grade bladder cancer and strongly correlates with a YAP/TAZ gene signature. These findings identify a positive feed forward loop in the Hippo pathway that establishes a key role for NUAK2 in enforcing the tumor-promoting activities of YAP/TAZ. Our results thus introduce a new opportunity for cancer therapeutics by delineating NUAK2 as a potential target for re-engaging the Hippo pathway.

PMID: 30158528 [PubMed - indexed for MEDLINE]



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Modeling the impact of drug interactions on therapeutic selectivity.

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Modeling the impact of drug interactions on therapeutic selectivity.

Nat Commun. 2018 08 27;9(1):3452

Authors: Weinstein ZB, Kuru N, Kiriakov S, Palmer AC, Khalil AS, Clemons PA, Zaman MH, Roth FP, Cokol M

Abstract
Combination therapies that produce synergistic growth inhibition are widely sought after for the pharmacotherapy of many pathological conditions. Therapeutic selectivity, however, depends on the difference between potency on disease-causing cells and potency on non-target cell types that cause toxic side effects. Here, we examine a model system of antimicrobial compound combinations applied to two highly diverged yeast species. We find that even though the drug interactions correlate between the two species, cell-type-specific differences in drug interactions are common and can dramatically alter the selectivity of compounds when applied in combination vs. single-drug activity-enhancing, diminishing, or inverting therapeutic windows. This study identifies drug combinations with enhanced cell-type-selectivity with a range of interaction types, which we experimentally validate using multiplexed drug-interaction assays for heterogeneous cell cultures. This analysis presents a model framework for evaluating drug combinations with increased efficacy and selectivity against pathogens or tumors.

PMID: 30150706 [PubMed - indexed for MEDLINE]



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Phage morons play important roles in controlling Pseudomonas aeruginosa phenotypes.

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Phage morons play important roles in controlling Pseudomonas aeruginosa phenotypes.

J Bacteriol. 2018 Aug 27;:

Authors: Tsao YF, Taylor VL, Kala S, Bondy-Denomy J, Khan AN, Bona D, Cattoir V, Lory S, Davidson AR, Maxwell KL

Abstract
The viruses that infect bacteria, known as phages, play critical roles in controlling bacterial populations in many diverse environments, including the human body. This control stems not only from phages killing bacteria, but also the formation of lysogens. In this state the phage replication cycle is suppressed, and the phage genome is maintained in the bacterial cell in a form known as a prophage. Prophages often carry genes that benefit the host bacterial cell since increasing survival of the host cell by extension also increases the fitness of the prophage. These highly diverse and beneficial phage genes, which are not required for the life cycle of the phage itself, have been referred to as "morons" as their presence adds "more on" the phage genome in which they are found. While individual phage morons have been shown to contribute to bacterial virulence by a number of different mechanisms, there have been no systematic investigations of their activities. Using a library of phages that infect two different clinical isolates of P. aeruginosa, PAO1 and PA14, we compared the phenotypes imparted by the expression of individual phage morons. We identified morons that inhibit twitching and swimming motilities and observed inhibition of production of virulence factors such as rhamnolipids and elastase. This study demonstrates the scope of phage-mediated phenotypic changes and provides a framework for future studies of phage morons.Importance Environmental and clinical isolates of the bacterium Pseudomonas aeruginosa frequently contain viruses known as prophages. These prophages can alter the virulence of their bacterial hosts through the expression of nonessential genes known as "morons". In this study we identified morons in a group of Pseudomonas aeruginosa phages and characterized the effects of their expression on bacterial behaviours. We found that many morons confer selective advantages for the bacterial host, some of which correlate with increased bacterial virulence. This work highlights the symbiotic relationship between bacteria and prophages and illustrates how phage morons can help bacteria adapt to different selective pressures and contribute to human diseases.

PMID: 30150232 [PubMed - as supplied by publisher]



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Essential Function of Mec1, the Budding Yeast ATM/ATR Checkpoint-Response Kinase, in Protein Homeostasis.

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Essential Function of Mec1, the Budding Yeast ATM/ATR Checkpoint-Response Kinase, in Protein Homeostasis.

Dev Cell. 2018 08 20;46(4):495-503.e2

Authors: Corcoles-Saez I, Dong K, Johnson AL, Waskiewicz E, Costanzo M, Boone C, Cha RS

Abstract
Unlike most checkpoint proteins, Mec1, an ATM/ATR kinase, is essential. We utilized mec1-4, a missense allele (E2130K) that confers diminished kinase activity, to interrogate the question. Unbiased screen for genetic interactors of mec1-4 identified numerous genes involved in proteostasis. mec1-4 confers sensitivity to heat, an amino acid analog, and Htt103Q, an aggregation-prone model peptide of Huntingtin. Oppositely, mec1-4 confers resistance to cycloheximide, a translation inhibitor. In response to heat, mec1-4 leads to widespread protein aggregation and cell death. Key components of the Mec1 signaling network, Rad53CHK1, Dun1, and Sml1, also impact survival in response to proteotoxic stress. Activation of autophagy or sml1Δ promotes aggregate resolution and rescues mec1-4 lethality. These findings show that proteostasis is a fundamental function of Mec1 and that Mec1 is likely to utilize its checkpoint response network to mediate resistance to proteotoxic stress, a role that may be conserved from yeast to mammalian cells.

PMID: 30130531 [PubMed - indexed for MEDLINE]



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SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue.

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SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue.

PLoS Pathog. 2018 Aug 20;14(8):e1007269

Authors: Kmiec D, Akbil B, Ananth S, Hotter D, Sparrer K, Stürzel CM, Trautz B, Ayouba A, Peeters M, Yao Z, Stagljar I, Passos V, Zillinger T, Goffinet C, Sauter D, Fackler OT, Kirchhoff F

Abstract
SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation.

PMID: 30125328 [PubMed - as supplied by publisher]



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The Banality of Anal: Safer Sexual Erotics in the Gay Men's Health Crisis' Safer Sex Comix and Ex Aequo's Alex et la vie d'après.

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The Banality of Anal: Safer Sexual Erotics in the Gay Men's Health Crisis' Safer Sex Comix and Ex Aequo's Alex et la vie d'après.

J Med Humanit. 2018 Aug 17;:

Authors: Greenblatt J

Abstract
Analyzing two harm reduction comics campaigns-one early in the AIDS crisis (the Gay Men's Health Crisis' [GMHC] 1980s Safer Sex Comix) and one more recent (Fabrice Neaud and Thierry Robberecht's 2008 Alex et la vie d'après), I explore tensions between queer safer sexual erotics and national discourses of sexual norms/deviation raised by Cindy Patton and William Haver at the height of AIDS discourse theory in 1996, approximately halfway between the comics. Using these theorists' reflections on the history of AIDS activism/representation as a hinge, I explore the manifestation/transformation a decade later of the ethical, educational, and erotic issues they raise. Both foreground the ways that HIV, safer sex, and/or eroticism pose difficulties for systems of linguistic and visual representation. Combining text and image, comics-a common harm reduction medium-epitomize this representational issue. While the GMHC addresses an immediate need for information about safer sex, Alex attempts to tackle the unrepresentability/unthinkability of safer and/or seropositive sex(uality). Safer Sex Comix, while largely prioritizing directness above formal experimentation, employ strategies of transgressing the borders of the comics panel to emphasize a plethora of lower-risk sexual acts. The most visually inventive moments in Alex represent Alex's feelings of unintelligbility post-diagnosis, but the comic restricts its representation of sex only to anal intercourse, and it proves unable to visualize alternative formulations of the erotic, turning to more normative narratives and images as earlier, visually explicit unsafe sexual encounters are replaced with more a/illusive representations post-conversion, literalizing the unrepresentability of seropositive erotic life.

PMID: 30120671 [PubMed - as supplied by publisher]



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