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Tracing the origins of relapse in acute myeloid leukaemia to stem cells.

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Tracing the origins of relapse in acute myeloid leukaemia to stem cells.

Nature. 2017 07 06;547(7661):104-108

Authors: Shlush LI, Mitchell A, Heisler L, Abelson S, Ng SWK, Trotman-Grant A, Medeiros JJF, Rao-Bhatia A, Jaciw-Zurakowsky I, Marke R, McLeod JL, Doedens M, Bader G, Voisin V, Xu C, McPherson JD, Hudson TJ, Wang JCY, Minden MD, Dick JE

Abstract
In acute myeloid leukaemia, long-term survival is poor as most patients relapse despite achieving remission. Historically, the failure of therapy has been thought to be due to mutations that produce drug resistance, possibly arising as a consequence of the mutagenic properties of chemotherapy drugs. However, other lines of evidence have pointed to the pre-existence of drug-resistant cells. For example, deep sequencing of paired diagnosis and relapse acute myeloid leukaemia samples has provided direct evidence that relapse in some cases is generated from minor genetic subclones present at diagnosis that survive chemotherapy, suggesting that resistant cells are generated by evolutionary processes before treatment and are selected by therapy. Nevertheless, the mechanisms of therapy failure and capacity for leukaemic regeneration remain obscure, as sequence analysis alone does not provide insight into the cell types that are fated to drive relapse. Although leukaemia stem cells have been linked to relapse owing to their dormancy and self-renewal properties, and leukaemia stem cell gene expression signatures are highly predictive of therapy failure, experimental studies have been primarily correlative and a role for leukaemia stem cells in acute myeloid leukaemia relapse has not been directly proved. Here, through combined genetic and functional analysis of purified subpopulations and xenografts from paired diagnosis/relapse samples, we identify therapy-resistant cells already present at diagnosis and two major patterns of relapse. In some cases, relapse originated from rare leukaemia stem cells with a haematopoietic stem/progenitor cell phenotype, while in other instances relapse developed from larger subclones of immunophenotypically committed leukaemia cells that retained strong stemness transcriptional signatures. The identification of distinct patterns of relapse should lead to improved methods for disease management and monitoring in acute myeloid leukaemia. Moreover, the shared functional and transcriptional stemness properties that underlie both cellular origins of relapse emphasize the importance of developing new therapeutic approaches that target stemness to prevent relapse.

PMID: 28658204 [PubMed - indexed for MEDLINE]



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Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens.

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Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens.

G3 (Bethesda). 2017 Jun 27;:

Authors: Hart T, Tong AHY, Chan K, Van Leeuwen J, Seetharaman A, Aregger M, Chandrashekhar M, Hustedt N, Seth S, Noonan A, Habsid A, Sizova O, Nedyalkova L, Climie R, Tworzyanski L, Lawson K, Sartori MA, Alibai S, Tieu D, Masud S, Mero P, Weiss A, Brown KR, Ušaj M, Billmann M, Rahman M, Costanzo M, Myers CL, Andrews BJ, Boone C, Durocher D, Moffat J

Abstract
The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we re-analyze 17 genome-scale knockout screens in human cell lines from three research groups using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference Core Essential Genes (CEG2) plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

PMID: 28655737 [PubMed - as supplied by publisher]



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RNAcompete-S: combined RNA sequence/structure preferences for RNA binding proteins derived from a single-step in vitro selection.

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RNAcompete-S: combined RNA sequence/structure preferences for RNA binding proteins derived from a single-step in vitro selection.

Methods. 2017 Jun 23;:

Authors: Cook KB, Vembu S, Ha KCH, Zheng H, Laverty KU, Hughes TR, Ray D, Morris QD

Abstract
RNA-binding proteins recognize RNA sequences and structures, but there is currently no systematic and accurate method to derive large (>12 base) motifs de novo that reflect a combination of intrinsic preference to both sequence and structure. To address this absence, we introduce RNAcompete-S, which couples a single-step competitive binding reaction with an excess of random RNA 40-mers to a custom computational pipeline for interrogation of the bound RNA sequences and derivation of SSMs (Sequence and Structure Models). RNAcompete-S confirms that HuR, QKI, and SRSF1 prefer binding sites that are single stranded, and recapitulates known 8-10 bp sequence and structure preferences for Vts1p and RBMY. We also derive an 18-base long SSM for Drosophila SLBP, which to our knowledge has not been previously determined by selections from pure random sequence, and accurately discriminates human replication-dependent histone mRNAs. Thus, RNAcompete-S enables accurate identification of large, intrinsic sequence-structure specificities with a uniform assay.

PMID: 28651966 [PubMed - as supplied by publisher]



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Recent advances in understanding contextual TGFβ signaling.

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Recent advances in understanding contextual TGFβ signaling.

F1000Res. 2017;6:749

Authors: Ayyaz A, Attisano L, Wrana JL

Abstract
The appearance of the first animal species on earth coincides with the emergence of transforming growth factor β (TGFβ) pathways. The evolution of these animals into more complex organisms coincides with a progressively increased TGFβ repertoire through gene duplications and divergence, making secreted TGFβ molecules the largest family of morphogenetic proteins in humans. It is therefore not surprising that TGFβ pathways govern numerous aspects of human biology from early embryonic development to regeneration, hematopoiesis, neurogenesis, and immunity. Such heavy reliance on these pathways is reflected in the susceptibility to minor perturbations in pathway components that can lead to dysregulated signaling and a diverse range of human pathologies such as cancer, fibrosis, and developmental disorders. Attempts to comprehensively resolve these signaling cascades are complicated by the long-recognized paradoxical role the pathway plays in cell biology. Recently, several groups have probed examples of the disparate aspects of TGFβ biology in a variety of animal models and uncovered novel context-dependent regulatory mechanisms. Here, we briefly review recent advancements and discuss their overall impact in directing future TGFβ research.

PMID: 28649369 [PubMed - in process]



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Effectively infinite optical path-length created using a simple cubic photonic crystal for extreme light trapping.

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Effectively infinite optical path-length created using a simple cubic photonic crystal for extreme light trapping.

Sci Rep. 2017 Jun 23;7(1):4171

Authors: Frey BJ, Kuang P, Hsieh ML, Jiang JH, John S, Lin SY

Abstract
A 900 nm thick TiO2 simple cubic photonic crystal with lattice constant 450 nm was fabricated and used to experimentally validate a newly-discovered mechanism for extreme light-bending. Absorption enhancement was observed extending 1-2 orders of magnitude over that of a reference TiO2 film. Several enhancement peaks in the region from 600-950 nm were identified, which far exceed both the ergodic fundamental limit and the limit based on surface-gratings, with some peaks exceeding 100 times enhancement. These results are attributed to radically sharp refraction where the optical path length approaches infinity due to the Poynting vector lying nearly parallel to the photonic crystal interface. The observed phenomena follow directly from the simple cubic symmetry of the photonic crystal, and can be achieved by integrating the light-trapping architecture into the absorbing volume. These results are not dependent on the material used, and can be applied to any future light trapping applications such as phosphor-converted white light generation, water-splitting, or thin-film solar cells, where increased response in areas of weak absorption is desired.

PMID: 28646167 [PubMed - in process]



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The phosphocarrier protein HPr of the bacterial phosphotransferase system globally regulates energy metabolism by directly interacting with multiple enzymes in Escherichia coli.

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The phosphocarrier protein HPr of the bacterial phosphotransferase system globally regulates energy metabolism by directly interacting with multiple enzymes in Escherichia coli.

J Biol Chem. 2017 Aug 25;292(34):14250-14257

Authors: Rodionova IA, Zhang Z, Mehla J, Goodacre N, Babu M, Emili A, Uetz P, Saier MH

Abstract
The histidine-phosphorylatable phosphocarrier protein (HPr) is an essential component of the sugar-transporting phosphotransferase system (PTS) in many bacteria. Recent interactome findings suggested that HPr interacts with several carbohydrate-metabolizing enzymes, but whether HPr plays a regulatory role was unclear. Here, we provide evidence that HPr interacts with a large number of proteins in Escherichia coli We demonstrate HPr-dependent allosteric regulation of the activities of pyruvate kinase (PykF, but not PykA), phosphofructokinase (PfkB, but not PfkA), glucosamine-6-phosphate deaminase (NagB), and adenylate kinase (Adk). HPr is either phosphorylated on a histidyl residue (HPr-P) or non-phosphorylated (HPr). PykF is activated only by non-phosphorylated HPr, which decreases the PykF Khalf for phosphoenolpyruvate by 10-fold (from 3.5 to 0.36 mm), thus influencing glycolysis. PfkB activation by HPr, but not by HPr-P, resulted from a decrease in the Khalf for fructose-6-P, which likely influences both gluconeogenesis and glycolysis. Moreover, NagB activation by HPr was important for the utilization of amino sugars, and allosteric inhibition of Adk activity by HPr-P, but not by HPr, allows HPr to regulate the cellular energy charge coordinately with glycolysis. These observations suggest that HPr serves as a directly interacting global regulator of carbon and energy metabolism and probably of other physiological processes in enteric bacteria.

PMID: 28634232 [PubMed - indexed for MEDLINE]



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Multiple functions of protein phosphatases in receptor tyrosine kinase signaling revealed by interactome analysis.

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Multiple functions of protein phosphatases in receptor tyrosine kinase signaling revealed by interactome analysis.

Mol Cell Oncol. 2017;4(3):e1297101

Authors: Yao Z, Stagljar I

Abstract
To obtain a global picture of how protein phosphatases are involved in receptor tyrosine kinase (RTK) signaling, we mapped the RTK-phosphatase interactome. Analyses of selected interactions revealed detailed mechanisms of their actions. This study provides new knowledge to better understand cancer development and to identify novel therapeutic targets.

PMID: 28616575 [PubMed - in process]



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Intertumoral Heterogeneity within Medulloblastoma Subgroups.

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Intertumoral Heterogeneity within Medulloblastoma Subgroups.

Cancer Cell. 2017 Jun 12;31(6):737-754.e6

Authors: Cavalli FMG, Remke M, Rampasek L, Peacock J, Shih DJH, Luu B, Garzia L, Torchia J, Nor C, Morrissy AS, Agnihotri S, Thompson YY, Kuzan-Fischer CM, Farooq H, Isaev K, Daniels C, Cho BK, Kim SK, Wang KC, Lee JY, Grajkowska WA, Perek-Polnik M, Vasiljevic A, Faure-Conter C, Jouvet A, Giannini C, Nageswara Rao AA, Li KKW, Ng HK, Eberhart CG, Pollack IF, Hamilton RL, Gillespie GY, Olson JM, Leary S, Weiss WA, Lach B, Chambless LB, Thompson RC, Cooper MK, Vibhakar R, Hauser P, van Veelen MC, Kros JM, French PJ, Ra YS, Kumabe T, López-Aguilar E, Zitterbart K, Sterba J, Finocchiaro G, Massimino M, Van Meir EG, Osuka S, Shofuda T, Klekner A, Zollo M, Leonard JR, Rubin JB, Jabado N, Albrecht S, Mora J, Van Meter TE, Jung S, Moore AS, Hallahan AR, Chan JA, Tirapelli DPC, Carlotti CG, Fouladi M, Pimentel J, Faria CC, Saad AG, Massimi L, Liau LM, Wheeler H, Nakamura H, Elbabaa SK, Perezpeña-Diazconti M, Chico Ponce de León F, Robinson S, Zapotocky M, Lassaletta A, Huang A, Hawkins CE, Tabori U, Bouffet E, Bartels U, Dirks PB, Rutka JT, Bader GD, Reimand J, Goldenberg A, Ramaswamy V, Taylor MD

Abstract
While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials.

PMID: 28609654 [PubMed - indexed for MEDLINE]



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Pre-concentration by liquid intake by paper (P-CLIP): a new technique for large volumes and digital microfluidics.

Pre-concentration by liquid intake by paper (P-CLIP): a new technique for large volumes and digital microfluidics.

Lab Chip. 2017 Jun 12;:

Authors: Rackus DG, de Campos RPS, Chan C, Karcz MM, Seale B, Narahari T, Dixon C, Chamberlain MD, Wheeler AR

Abstract
Microfluidic platforms are an attractive option for incorporating complex fluid handling into low-cost and rapid diagnostic tests. A persistent challenge for microfluidics, however, is the mismatch in the "world-to-chip" interface - it is challenging to detect analytes present at low concentrations in systems that can only handle small volumes of sample. Here we describe a new technique termed pre-concentration by liquid intake by paper (P-CLIP) that addresses this mismatch, allowing digital microfluidics to interface with volumes on the order of hundreds of microliters. In P-CLIP, a virtual microchannel is generated to pass a large volume through the device; analytes captured on magnetic particles can be isolated and then resuspended into smaller volumes for further processing and analysis. We characterize this method and demonstrate its utility with an immunoassay for Plasmodium falciparum lactate dehydrogenase, a malaria biomarker, and propose that the P-CLIP strategy may be useful for a wide range of applications that are currently limited by low-abundance analytes.

PMID: 28604891 [PubMed - as supplied by publisher]



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Rab7 palmitoylation is required for efficient endosome-to-TGN trafficking.

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Rab7 palmitoylation is required for efficient endosome-to-TGN trafficking.

J Cell Sci. 2017 Jun 09;:

Authors: Modica G, Skorobogata O, Sauvageau E, Vissa A, Yip CM, Kim PK, Wurtele H, Lefrancois S

Abstract
Retromer is a multimeric protein complex that mediates endosome-to-TGN and endosome-to-plasma membrane trafficking of integral membrane proteins. Dysfunction of this complex has been linked to Alzheimer's and Parkinson's disease. The recruitment of retromer to endosomes is regulated by Rab7 to coordinate endosome-to- TGN trafficking of cargo-receptor complexes. Rab7 is also required for the degradation of internalized integral membrane proteins such as the epidermal growth factor receptor. We found that Rab7 is palmitoylated and that this modification is not required for membrane anchoring. Palmitoylated Rab7 co-localizes efficiently with and has a higher propensity to interact with retromer than non-palmitoylatable Rab7. Rescue of Rab7 knock out cells by expressing wild-type Rab7 restores efficient endosome-to-TGN trafficking, while rescue with non-palmitoylatable Rab7 does not. Interestingly, Rab7 palmitoylation does not appear to be required for the degradation of epidermal growth factor receptor receptor nor its interaction with its effector RILP. Overall, our results indicate that Rab7 palmitoylation is required for the spatiotemporal recruitment of retromer and efficient endosome-to-TGN Network trafficking of the lysosomal sorting receptors.

PMID: 28600323 [PubMed - as supplied by publisher]



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