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CD200 expression marks leukemia stem cells in human AML.

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CD200 expression marks leukemia stem cells in human AML.

Blood Adv. 2020 Nov 10;4(21):5402-5413

Authors: Ho JM, Dobson SM, Voisin V, McLeod J, Kennedy JA, Mitchell A, Jin L, Eppert K, Bader G, Minden MD, Dick JE, Wang JCY

Abstract
The leukemia stem cell (LSC) populations of acute myeloid leukemia (AML) exhibit phenotypic, genetic, and functional heterogeneity that contribute to therapy failure and relapse. Progress toward understanding the mechanistic basis for therapy resistance in LSCs has been hampered by difficulties in isolating cell fractions that enrich for the entire heterogeneous population of LSCs within individual AML samples. We previously reported that CD200 gene expression is upregulated in LSC-containing AML fractions. Here, we show that CD200 is present on a greater proportion of CD45dim blasts compared with more differentiated CD45high cells in AML patient samples. In 75% (49 of 65) of AML cases we examined, CD200 was expressed on ≥10% of CD45dim blasts; of these, CD200 identified LSCs within the blast population in 9 of 10 (90%) samples tested in xenotransplantation assays. CD200+ LSCs could be isolated from CD200+ normal HSCs with the use of additional markers. Notably, CD200 expression captured both CD34- and CD34+ LSCs within individual AML samples. Analysis of highly purified CD200+ LSC-containing fractions from NPM1-mutated AMLs, which are commonly CD34-, exhibited an enrichment of primitive gene expression signatures compared with unfractionated cells. Overall, our findings support CD200 as a novel LSC marker that is able to capture the entire LSC compartment from AML patient samples, including those with NPM1 mutation.

PMID: 33147339 [PubMed - as supplied by publisher]



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Genetic profiling of protein burden and nuclear export overload.

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Genetic profiling of protein burden and nuclear export overload.

Elife. 2020 Nov 04;9:

Authors: Kintaka R, Makanae K, Namba S, Kato H, Kito K, Ohnuki S, Ohya Y, Andrews BJ, Boone C, Moriya H

Abstract
Overproduction (op) of proteins triggers cellular defects. One of the consequences of overproduction is the protein burden/cost, which is produced by an overloading of the protein synthesis process. However, the physiology of cells under a protein burden is not well characterized. We performed genetic profiling of protein burden by systematic analysis of genetic interactions between GFP-op, surveying both deletion and temperature-sensitive mutants in budding yeast. We also performed genetic profiling in cells with overproduction of triple-GFP (tGFP), and the nuclear export signal-containing tGFP (NES-tGFP). The mutants specifically interacted with GFP-op were suggestive of unexpected connections between actin-related processes like polarization and the protein burden, which was supported by morphological analysis. The tGFP-op interactions suggested that this protein probe overloads the proteasome, whereas those that interacted with NES-tGFP involved genes encoding components of the nuclear export process, providing a resource for further analysis of the protein burden and nuclear export overload.

PMID: 33146608 [PubMed - as supplied by publisher]



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Intensive Care Unit-Acquired Weakness: Not just Another Muscle Atrophying Condition.

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Intensive Care Unit-Acquired Weakness: Not just Another Muscle Atrophying Condition.

Int J Mol Sci. 2020 Oct 22;21(21):

Authors: Lad H, Saumur TM, Herridge MS, Dos Santos CC, Mathur S, Batt J, Gilbert PM

Abstract
Intensive care unit-acquired weakness (ICUAW) occurs in critically ill patients stemming from the critical illness itself, and results in sustained disability long after the ICU stay. Weakness can be attributed to muscle wasting, impaired contractility, neuropathy, and major pathways associated with muscle protein degradation such as the ubiquitin proteasome system and dysregulated autophagy. Furthermore, it is characterized by the preferential loss of myosin, a distinct feature of the condition. While many risk factors for ICUAW have been identified, effective interventions to offset these changes remain elusive. In addition, our understanding of the mechanisms underlying the long-term, sustained weakness observed in a subset of patients after discharge is minimal. Herein, we discuss the various proposed pathways involved in the pathophysiology of ICUAW, with a focus on the mechanisms underpinning skeletal muscle wasting and impaired contractility, and the animal models used to study them. Furthermore, we will explore the contributions of inflammation, steroid use, and paralysis to the development of ICUAW and how it pertains to those with the corona virus disease of 2019 (COVID-19). We then elaborate on interventions tested as a means to offset these decrements in muscle function that occur as a result of critical illness, and we propose new strategies to explore the molecular mechanisms of ICUAW, including serum-related biomarkers and 3D human skeletal muscle culture models.

PMID: 33105809 [PubMed - indexed for MEDLINE]



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TPR is required for the efficient nuclear export of mRNAs and lncRNAs from short and intron-poor genes.

TPR is required for the efficient nuclear export of mRNAs and lncRNAs from short and intron-poor genes.

Nucleic Acids Res. 2020 Oct 22;:

Authors: Lee ES, Wolf EJ, Ihn SSJ, Smith HW, Emili A, Palazzo AF

Abstract
While splicing has been shown to enhance nuclear export, it has remained unclear whether mRNAs generated from intronless genes use specific machinery to promote their export. Here, we investigate the role of the major nuclear pore basket protein, TPR, in regulating mRNA and lncRNA nuclear export in human cells. By sequencing mRNA from the nucleus and cytosol of control and TPR-depleted cells, we provide evidence that TPR is required for the efficient nuclear export of mRNAs and lncRNAs that are generated from short transcripts that tend to have few introns, and we validate this with reporter constructs. Moreover, in TPR-depleted cells reporter mRNAs generated from short transcripts accumulate in nuclear speckles and are bound to Nxf1. These observations suggest that TPR acts downstream of Nxf1 recruitment and may allow mRNAs to leave nuclear speckles and properly dock with the nuclear pore. In summary, our study provides one of the first examples of a factor that is specifically required for the nuclear export of intronless and intron-poor mRNAs and lncRNAs.

PMID: 33091126 [PubMed - as supplied by publisher]



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Chemical-Genetic Interactions with the Proline Analog L-Azetidine-2-Carboxylic Acid in Saccharomyces cerevisiae.

Chemical-Genetic Interactions with the Proline Analog L-Azetidine-2-Carboxylic Acid in Saccharomyces cerevisiae.

G3 (Bethesda). 2020 Oct 20;:

Authors: Berg MD, Zhu Y, Isaacson J, Genereaux J, Loll-Krippleber R, Brown GW, Brandl CJ

Abstract
Non-proteinogenic amino acids, such as the proline analog L-azetidine-2-carboxylic acid (AZC), are detrimental to cells because they are mis-incorporated into proteins and lead to proteotoxic stress. Our goal was to identify genes that show chemical-genetic interactions with AZC in Saccharomyces cerevisiae and thus also potentially define the pathways cells use to cope with amino acid mis-incorporation. Screening the yeast deletion and temperature sensitive collections, we found 72 alleles with negative chemical-genetic interactions with AZC treatment and 12 alleles that suppress AZC toxicity. Many of the genes with negative chemical-genetic interactions are involved in protein quality control pathways through the proteasome. Genes involved in actin cytoskeleton organization and endocytosis also had negative chemical-genetic interactions with AZC. Related to this, the number of actin patches per cell increases upon AZC treatment. Many of the same cellular processes were identified to have interactions with proteotoxic stress caused by two other amino acid analogs, canavanine and thialysine, or a mistranslating tRNA variant that mis-incorporates serine at proline codons. Alleles that suppressed AZC-induced toxicity functioned through the amino acid sensing TOR pathway or controlled amino acid permeases required for AZC uptake. Further suggesting the potential of genetic changes to influence the cellular response to proteotoxic stress, overexpressing many of the genes that had a negative chemical-genetic interaction with AZC suppressed AZC toxicity.

PMID: 33082270 [PubMed - as supplied by publisher]



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The structure and function of deubiquitinases: lessons from budding yeast.

The structure and function of deubiquitinases: lessons from budding yeast.

Open Biol. 2020 Oct;10(10):200279

Authors: Suresh HG, Pascoe N, Andrews B

Abstract
Protein ubiquitination is a key post-translational modification that regulates diverse cellular processes in eukaryotic cells. The specificity of ubiquitin (Ub) signalling for different bioprocesses and pathways is dictated by the large variety of mono-ubiquitination and polyubiquitination events, including many possible chain architectures. Deubiquitinases (DUBs) reverse or edit Ub signals with high sophistication and specificity, forming an integral arm of the Ub signalling machinery, thus impinging on fundamental cellular processes including DNA damage repair, gene expression, protein quality control and organellar integrity. In this review, we discuss the many layers of DUB function and regulation, with a focus on insights gained from budding yeast. Our review provides a framework to understand key aspects of DUB biology.

PMID: 33081638 [PubMed - in process]



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The GATOR-Rag GTPase pathway inhibits mTORC1 activation by lysosome-derived amino acids.

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The GATOR-Rag GTPase pathway inhibits mTORC1 activation by lysosome-derived amino acids.

Science. 2020 10 16;370(6514):351-356

Authors: Hesketh GG, Papazotos F, Pawling J, Rajendran D, Knight JDR, Martinez S, Taipale M, Schramek D, Dennis JW, Gingras AC

Abstract
The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR-Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase-independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.

PMID: 33060361 [PubMed - indexed for MEDLINE]



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Interrogation of kinase genetic interactions provides a global view of PAK1-mediated signal transduction pathways.

Interrogation of kinase genetic interactions provides a global view of PAK1-mediated signal transduction pathways.

J Biol Chem. 2020 Oct 15;:

Authors: Kim JH, Seo Y, Jo M, Jeon H, Kim YS, Kim EJ, Seo D, Lee WH, Kim SR, Yachie N, Zhong Q, Vidal M, Roth FP, Suk K

Abstract
Kinases are critical components of intracellular signaling pathways and have been extensively investigated in regards to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid S. cerevisiae yeast deletion mutants identified approximately 400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration, and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy.

PMID: 33060198 [PubMed - as supplied by publisher]



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An Injectable Hyaluronan-Methylcellulose (HAMC) Hydrogel Combined with Wharton's Jelly-Derived Mesenchymal Stromal Cells (WJ-MSCs) Promotes Degenerative Disc Repair.

An Injectable Hyaluronan-Methylcellulose (HAMC) Hydrogel Combined with Wharton's Jelly-Derived Mesenchymal Stromal Cells (WJ-MSCs) Promotes Degenerative Disc Repair.

Int J Mol Sci. 2020 Oct 07;21(19):

Authors: Choi UY, Joshi HP, Payne S, Kim KT, Kyung JW, Choi H, Cooke MJ, Kwon SY, Roh EJ, Sohn S, Shoichet MS, Han I

Abstract
Intervertebral disc (IVD) degeneration is one of the predominant causes of chronic low back pain (LBP), which is a leading cause of disability worldwide. Despite substantial progress in cell therapy for the treatment of IVD degeneration, significant challenges remain for clinical application. Here, we investigated the effectiveness of hyaluronan-methylcellulose (HAMC) hydrogels loaded with Wharton's Jelly-derived mesenchymal stromal cell (WJ-MSCs) in vitro and in a rat coccygeal IVD degeneration model. Following induction of injury-induced IVD degeneration, female Sprague-Dawley rats were randomized into four groups to undergo a single intradiscal injection of the following: (1) phosphate buffered saline (PBS) vehicle, (2) HAMC, (3) WJ-MSCs (2 × 104 cells), and (4) WJ-MSCs-loaded HAMC (WJ-MSCs/HAMC) (n = 10/each group). Coccygeal discs were removed following sacrifice 6 weeks after implantation for radiologic and histologic analysis. We confirmed previous findings that encapsulation in HAMC increases the viability of WJ-MSCs for disc repair. The HAMC gel maintained significant cell viability in vitro. In addition, combined implantation of WJ-MSCs and HAMC significantly promoted degenerative disc repair compared to WJ-MSCs alone, presumably by improving nucleus pulposus cells viability and decreasing extracellular matrix degradation. Our results suggest that WJ-MSCs-loaded HAMC promotes IVD repair more effectively than cell injection alone and supports the potential clinical use of HAMC for cell delivery to arrest IVD degeneration or to promote IVD regeneration.

PMID: 33036383 [PubMed - as supplied by publisher]



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DNA-Controlled Encapsulation of Small Molecules in Protein Nanoparticles.

DNA-Controlled Encapsulation of Small Molecules in Protein Nanoparticles.

J Am Chem Soc. 2020 Oct 06;:

Authors: Ngo W, Stordy B, Lazarovits J, Raja EK, Etienne CL, Chan WCW

Abstract
A nanoparticle can hold multiple types of therapeutic and imaging agents for disease treatment and diagnosis. However, controlling the storage of molecules in nanoparticles is challenging, because nonspecific intermolecular interactions are used for encapsulation. Here, we used specific DNA interactions to store molecules in nanoparticles. We made nanoparticles containing DNA anchors to capture DNA-conjugated small molecules. By changing the sequences and stoichiometry of DNA anchors, we can control the amount and ratio of molecules with different chemical properties in the nanoparticles. We modified the cytotoxicity of our nanoparticles to cancer cells by changing the ratio of encapsulated drugs (mertansine and doxorubicin). Specifically controlling the storage of multiple types of molecules allows us to optimize the properties of combination drug and imaging nanoparticles.

PMID: 33022172 [PubMed - as supplied by publisher]



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