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MKRN2 Physically Interacts with GLE1 to Regulate mRNA Export and Zebrafish Retinal Development.

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MKRN2 Physically Interacts with GLE1 to Regulate mRNA Export and Zebrafish Retinal Development.

Cell Rep. 2020 May 26;31(8):107693

Authors: Wolf EJ, Miles A, Lee ES, Nabeel-Shah S, Greenblatt JF, Palazzo AF, Tropepe V, Emili A

Abstract
The mammalian mRNA nuclear export process is thought to terminate at the cytoplasmic face of the nuclear pore complex through ribonucleoprotein remodeling. We conduct a stringent affinity-purification mass-spectrometry-based screen of the physical interactions of human RNA-binding E3 ubiquitin ligases. The resulting protein-interaction network reveals interactions between the RNA-binding E3 ubiquitin ligase MKRN2 and GLE1, a DEAD-box helicase activator implicated in mRNA export termination. We assess MKRN2 epistasis with GLE1 in a zebrafish model. Morpholino-mediated knockdown or CRISPR/Cas9-based knockout of MKRN2 partially rescue retinal developmental defects seen upon GLE1 depletion, consistent with a functional association between GLE1 and MKRN2. Using ribonomic approaches, we show that MKRN2 binds selectively to the 3' UTR of a diverse subset of mRNAs and that nuclear export of MKRN2-associated mRNAs is enhanced upon knockdown of MKRN2. Taken together, we suggest that MKRN2 interacts with GLE1 to selectively regulate mRNA nuclear export and retinal development.

PMID: 32460013 [PubMed - as supplied by publisher]



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Distinct fibroblast functional states drive clinical outcomes in ovarian cancer and are regulated by TCF21.

Distinct fibroblast functional states drive clinical outcomes in ovarian cancer and are regulated by TCF21.

J Exp Med. 2020 Aug 03;217(8):

Authors: Hussain A, Voisin V, Poon S, Karamboulas C, Bui NHB, Meens J, Dmytryshyn J, Ho VW, Tang KH, Paterson J, Clarke BA, Bernardini MQ, Bader GD, Neel BG, Ailles LE

Abstract
Recent studies indicate that cancer-associated fibroblasts (CAFs) are phenotypically and functionally heterogeneous. However, little is known about CAF subtypes, the roles they play in cancer progression, and molecular mediators of the CAF "state." Here, we identify a novel cell surface pan-CAF marker, CD49e, and demonstrate that two distinct CAF states, distinguished by expression of fibroblast activation protein (FAP), coexist within the CD49e+ CAF compartment in high-grade serous ovarian cancers. We show for the first time that CAF state influences patient outcomes and that this is mediated by the ability of FAP-high, but not FAP-low, CAFs to aggressively promote proliferation, invasion and therapy resistance of cancer cells. Overexpression of the FAP-low-specific transcription factor TCF21 in FAP-high CAFs decreases their ability to promote invasion, chemoresistance, and in vivo tumor growth, indicating that it acts as a master regulator of the CAF state. Understanding CAF states in more detail could lead to better patient stratification and novel therapeutic strategies.

PMID: 32434219 [PubMed - as supplied by publisher]



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Amino acid and lipid metabolism in post-gestational diabetes and progression to type 2 diabetes: A metabolic profiling study.

Amino acid and lipid metabolism in post-gestational diabetes and progression to type 2 diabetes: A metabolic profiling study.

PLoS Med. 2020 May;17(5):e1003112

Authors: Lai M, Liu Y, Ronnett GV, Wu A, Cox BJ, Dai FF, Röst HL, Gunderson EP, Wheeler MB

Abstract
BACKGROUND: Women with a history of gestational diabetes mellitus (GDM) have a 7-fold higher risk of developing type 2 diabetes (T2D) during midlife and an elevated risk of developing hypertension and cardiovascular disease. Glucose tolerance reclassification after delivery is recommended, but fewer than 40% of women with GDM are tested. Thus, improved risk stratification methods are needed, as is a deeper understanding of the pathology underlying the transition from GDM to T2D. We hypothesize that metabolites during the early postpartum period accurately distinguish risk of progression from GDM to T2D and that metabolite changes signify underlying pathophysiology for future disease development.
METHODS AND FINDINGS: The study utilized fasting plasma samples collected from a well-characterized prospective research study of 1,035 women diagnosed with GDM. The cohort included racially/ethnically diverse pregnant women (aged 20-45 years-33% primiparous, 37% biparous, 30% multiparous) who delivered at Kaiser Permanente Northern California hospitals from 2008 to 2011. Participants attended in-person research visits including 2-hour 75-g oral glucose tolerance tests (OGTTs) at study baseline (6-9 weeks postpartum) and annually thereafter for 2 years, and we retrieved diabetes diagnoses from electronic medical records for 8 years. In a nested case-control study design, we collected fasting plasma samples among women without diabetes at baseline (n = 1,010) to measure metabolites among those who later progressed to incident T2D or did not develop T2D (non-T2D). We studied 173 incident T2D cases and 485 controls (pair-matched on BMI, age, and race/ethnicity) to discover metabolites associated with new onset of T2D. Up to 2 years post-baseline, we analyzed samples from 98 T2D cases with 239 controls to reveal T2D-associated metabolic changes. The longitudinal analysis tracked metabolic changes within individuals from baseline to 2 years of follow-up as the trajectory of T2D progression. By building prediction models, we discovered a distinct metabolic signature in the early postpartum period that predicted future T2D with a median discriminating power area under the receiver operating characteristic curve of 0.883 (95% CI 0.820-0.945, p < 0.001). At baseline, the most striking finding was an overall increase in amino acids (AAs) as well as diacyl-glycerophospholipids and a decrease in sphingolipids and acyl-alkyl-glycerophospholipids among women with incident T2D. Pathway analysis revealed up-regulated AA metabolism, arginine/proline metabolism, and branched-chain AA (BCAA) metabolism at baseline. At follow-up after the onset of T2D, up-regulation of AAs and down-regulation of sphingolipids and acyl-alkyl-glycerophospholipids were sustained or strengthened. Notably, longitudinal analyses revealed only 10 metabolites associated with progression to T2D, implicating AA and phospholipid metabolism. A study limitation is that all of the analyses were performed with the same cohort. It would be ideal to validate our findings in an independent longitudinal cohort of women with GDM who had glucose tolerance tested during the early postpartum period.
CONCLUSIONS: In this study, we discovered a metabolic signature predicting the transition from GDM to T2D in the early postpartum period that was superior to clinical parameters (fasting plasma glucose, 2-hour plasma glucose). The findings suggest that metabolic dysregulation, particularly AA dysmetabolism, is present years prior to diabetes onset, and is revealed during the early postpartum period, preceding progression to T2D, among women with GDM.
TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01967030.

PMID: 32433647 [PubMed - as supplied by publisher]



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Disrupting Mitochondrial Copper Distribution Inhibits Leukemic Stem Cell Self-Renewal.

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Disrupting Mitochondrial Copper Distribution Inhibits Leukemic Stem Cell Self-Renewal.

Cell Stem Cell. 2020 May 12;:

Authors: Singh RP, Jeyaraju DV, Voisin V, Hurren R, Xu C, Hawley JR, Barghout SH, Khan DH, Gronda M, Wang X, Jitkova Y, Sharon D, Liyanagae S, MacLean N, Seneviratene AK, Mirali S, Borenstein A, Thomas GE, Soriano J, Orouji E, Minden MD, Arruda A, Chan SM, Bader GD, Lupien M, Schimmer AD

Abstract
Leukemic stem cells (LSCs) rely on oxidative metabolism and are differentially sensitive to targeting mitochondrial pathways, which spares normal hematopoietic cells. A subset of mitochondrial proteins is folded in the intermembrane space via the mitochondrial intermembrane assembly (MIA) pathway. We found increased mRNA expression of MIA pathway substrates in acute myeloid leukemia (AML) stem cells. Therefore, we evaluated the effects of inhibiting this pathway in AML. Genetic and chemical inhibition of ALR reduces AML growth and viability, disrupts LSC self-renewal, and induces their differentiation. ALR inhibition preferentially decreases its substrate COX17, a mitochondrial copper chaperone, and knockdown of COX17 phenocopies ALR loss. Inhibiting ALR and COX17 increases mitochondrial copper levels which in turn inhibit S-adenosylhomocysteine hydrolase (SAHH) and lower levels of S-adenosylmethionine (SAM), DNA methylation, and chromatin accessibility to lower LSC viability. These results provide insight into mechanisms through which mitochondrial copper controls epigenetic status and viability of LSCs.

PMID: 32416059 [PubMed - as supplied by publisher]



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Split Intein-Mediated Protein Ligation for detecting protein-protein interactions and their inhibition.

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Split Intein-Mediated Protein Ligation for detecting protein-protein interactions and their inhibition.

Nat Commun. 2020 May 15;11(1):2440

Authors: Yao Z, Aboualizadeh F, Kroll J, Akula I, Snider J, Lyakisheva A, Tang P, Kotlyar M, Jurisica I, Boxem M, Stagljar I

Abstract
Here, to overcome many limitations accompanying current available methods to detect protein-protein interactions (PPIs), we develop a live cell method called Split Intein-Mediated Protein Ligation (SIMPL). In this approach, bait and prey proteins are respectively fused to an intein N-terminal fragment (IN) and C-terminal fragment (IC) derived from a re-engineered split intein GP41-1. The bait/prey binding reconstitutes the intein, which splices the bait and prey peptides into a single intact protein that can be detected by regular protein detection methods such as Western blot analysis and ELISA, serving as readouts of PPIs. The method is robust and can be applied not only in mammalian cell lines but in animal models such as C. elegans. SIMPL demonstrates high sensitivity and specificity, and enables exploration of PPIs in different cellular compartments and tracking of kinetic interactions. Additionally, we establish a SIMPL ELISA platform that enables high-throughput screening of PPIs and their inhibitors.

PMID: 32415080 [PubMed - as supplied by publisher]



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APOE ε4 and the influence of sex, age, vascular risk factors, and ethnicity on cognitive decline.

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APOE ε4 and the influence of sex, age, vascular risk factors, and ethnicity on cognitive decline.

J Gerontol A Biol Sci Med Sci. 2020 May 12;:

Authors: Makkar SR, Lipnicki DM, Crawford JD, Kochan NA, Castro-Costa E, Lima-Costa MF, Diniz BS, Brayne C, Stephan B, Matthews F, Llibre-Rodriguez JJ, Llibre-Guerra JJ, Valhuerdi-Cepero AJ, Lipton RB, Katz MJ, Wang C, Ritchie K, Carles S, Carriere I, Scarmeas N, Yannakoulia M, Kosmidis M, Lam L, Chan WC, Fung A, Guaita A, Vaccaro R, Davin A, Kim KW, Han JW, Suh SW, Riedel-Heller SG, Roehr S, Pabst A, Ganguli M, Hughes TF, Snitz B, Anstey KJ, Cherbuin N, Easteal S, Haan MN, Aiello AE, Dang K, Ng TP, Gao Q, Nyunt MSZ, Brodaty H, Trollor JN, Leung Y, Lo JW, Sachdev P, for Cohort Studies of Memory in an International Consortium (COSMIC)

Abstract
We aimed to examine the relationship between APOE*4 carriage on cognitive decline, and whether these associations were moderated by sex, baseline age, ethnicity, and vascular risk factors. Participants were 19,225 individuals aged 54-103 years from 15 longitudinal cohort studies with a mean follow up duration ranging between 1.2 and 10.7 years. Two-step individual participant data (IPD) meta-analysis was used to pool results of study-wise analyses predicting memory and general cognitive decline from carriage of one or two APOE*4 alleles, and moderation of these associations by age, sex, vascular risk factors and ethnicity. Separate pooled estimates were calculated in both men and women who were younger (i.e., 62 years) and older (i.e., 80 years) at baseline. Results showed that APOE*4 carriage was related to faster general cognitive decline in women, and faster memory decline in men. A stronger dose-dependent effect was observed in older men, with faster general cognitive and memory decline in those carrying two versus one APOE*4 allele. Vascular risk factors were related to an increased effect of APOE*4 on memory decline in younger women, but a weaker effect of APOE*4 on general cognitive decline in older men. The relationship between APOE*4 carriage and memory decline was larger in older-aged Asians than Whites. In sum, APOE*4 is related to cognitive decline in men and women, although these effects are enhanced by age and carriage of two APOE*4 alleles in men, a higher numbers of vascular risk factors during the early stages of late adulthood in women, and Asian ethnicity.

PMID: 32396611 [PubMed - as supplied by publisher]



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Functional culture and in vitro genetic and small-molecule manipulation of adult mouse cardiomyocytes.

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Functional culture and in vitro genetic and small-molecule manipulation of adult mouse cardiomyocytes.

Commun Biol. 2020 May 11;3(1):229

Authors: Callaghan NI, Lee SH, Hadipour-Lakmehsari S, Lee XA, Ahsan Siraj M, Driouchi A, Yip CM, Husain M, Simmons CA, Gramolini AO

Abstract
Primary adult cardiomyocyte (aCM) represent the mature form of myocytes found in the adult heart. However, culture of aCMs in particular is challenged by poor survival and loss of phenotype, rendering extended in vitro experiments unfeasible. Here, we establish murine aCM culture methods that enhance survival and maintain sarcomeric structure and Ca2+ cycling to enable physiologically relevant contractile force measurements. We also demonstrate genetic and small-molecule manipulations that probe mechanisms underlying myocyte functional performance. Together, these refinements to aCM culture present a toolbox with which to advance our understanding of myocardial physiology.

PMID: 32393743 [PubMed - in process]



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Yeast-Based Genetic Interaction Analysis of Human Kinome.

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Yeast-Based Genetic Interaction Analysis of Human Kinome.

Cells. 2020 May 07;9(5):

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

Abstract
Kinases are critical intracellular signaling proteins. To better understand kinase-mediated signal transduction, a large-scale human-yeast genetic interaction screen was performed. Among 597 human kinase genes tested, 28 displayed strong toxicity in yeast when overexpressed. En masse transformation of these toxic kinase genes into 4653 homozygous diploid yeast deletion mutants followed by barcode sequencing identified yeast toxicity modifiers and thus their human orthologs. Subsequent network analyses and functional grouping revealed that the 28 kinases and their 676 interaction partners (corresponding to a total of 969 genetic interactions) are enriched in cell death and survival (34%), small-molecule biochemistry (18%) and molecular transport (11%), among others. In the subnetwork analyses, a few kinases were commonly associated with glioma, cell migration and cell death/survival. Our analysis enabled the creation of a first draft of the kinase genetic interactome network and identified multiple drug targets for inflammatory diseases and cancer, in which deregulated kinase signaling plays a pathogenic role.

PMID: 32392905 [PubMed - in process]



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The geometric influence on the Cys2His2 zinc finger domain and functional plasticity.

The geometric influence on the Cys2His2 zinc finger domain and functional plasticity.

Nucleic Acids Res. 2020 May 08;:

Authors: Mueller AL, Corbi-Verge C, Giganti DO, Ichikawa DM, Spencer JM, MacRae M, Garton M, Kim PM, Noyes MB

Abstract
The Cys2His2 zinc finger is the most common DNA-binding domain expanding in metazoans since the fungi human split. A proposed catalyst for this expansion is an arms race to silence transposable elements yet it remains poorly understood how this domain is able to evolve the required specificities. Likewise, models of its DNA binding specificity remain error prone due to a lack of understanding of how adjacent fingers influence each other's binding specificity. Here, we use a synthetic approach to exhaustively investigate binding geometry, one of the dominant influences on adjacent finger function. By screening over 28 billion protein-DNA interactions in various geometric contexts we find the plasticity of the most common natural geometry enables more functional amino acid combinations across all targets. Further, residues that define this geometry are enriched in genomes where zinc fingers are prevalent and specificity transitions would be limited in alternative geometries. Finally, these results demonstrate an exhaustive synthetic screen can produce an accurate model of domain function while providing mechanistic insight that may have assisted in the domains expansion.

PMID: 32383734 [PubMed - as supplied by publisher]



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Flow Rate Affects Nanoparticle Uptake into Endothelial Cells.

Flow Rate Affects Nanoparticle Uptake into Endothelial Cells.

Adv Mater. 2020 May 08;:e1906274

Authors: Chen YY, Syed AM, MacMillan P, Rocheleau JV, Chan WCW

Abstract
Nanoparticles are commonly administered through systemic injection, which exposes them to the dynamic environment of the bloodstream. Injected nanoparticles travel within the blood and experience a wide range of flow velocities that induce varying shear rates to the blood vessels. Endothelial cells line these vessels, and have been shown to uptake nanoparticles during circulation, but it is difficult to characterize the flow-dependence of this interaction in vivo. Here, a microfluidic system is developed to control the flow rates of nanoparticles as they interact with endothelial cells. Gold nanoparticle uptake into endothelial cells is quantified at varying flow rates, and it is found that increased flow rates lead to decreased nanoparticle uptake. Endothelial cells respond to increased flow shear with decreased ability to uptake the nanoparticles. If cells are sheared the same way, nanoparticle uptake decreases as their flow velocity increases. Modifying nanoparticle surfaces with endothelial-cell-binding ligands partially restores uptake to nonflow levels, suggesting that functionalizing nanoparticles to bind to endothelial cells enables nanoparticles to resist flow effects. In the future, this microfluidic system can be used to test other nanoparticle-endothelial cell interactions under flow. The results of these studies can guide the engineering of nanoparticles for in vivo medical applications.

PMID: 32383233 [PubMed - as supplied by publisher]



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