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Attenuated diphtheria toxin mediates siRNA delivery.

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Attenuated diphtheria toxin mediates siRNA delivery.

Sci Adv. 2020 May;6(18):eaaz4848

Authors: Arnold AE, Smith LJ, Beilhartz G, Bahlmann LC, Jameson E, Melnyk R, Shoichet MS

Abstract
Toxins efficiently deliver cargo to cells by binding to cell surface ligands, initiating endocytosis, and escaping the endolysosomal pathway into the cytoplasm. We took advantage of this delivery pathway by conjugating an attenuated diphtheria toxin to siRNA, thereby achieving gene downregulation in patient-derived glioblastoma cells. We delivered siRNA against integrin-β1 (ITGB1)-a gene that promotes invasion and metastasis-and siRNA against eukaryotic translation initiation factor 3 subunit b (eIF-3b)-a survival gene. We demonstrated mRNA downregulation of both genes and the corresponding functional outcomes: knockdown of ITGB1 led to a significant inhibition of invasion, shown with an innovative 3D hydrogel model; and knockdown of eIF-3b resulted in significant cell death. This is the first example of diphtheria toxin being used to deliver siRNAs, and the first time a toxin-based siRNA delivery strategy has been shown to induce relevant genotypic and phenotypic effects in cancer cells.

PMID: 32494676 [PubMed - in process]



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A Dynamic Splicing Program Ensures Proper Synaptic Connections in the Developing Cerebellum.

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A Dynamic Splicing Program Ensures Proper Synaptic Connections in the Developing Cerebellum.

Cell Rep. 2020 Jun 02;31(9):107703

Authors: Farini D, Cesari E, Weatheritt RJ, La Sala G, Naro C, Pagliarini V, Bonvissuto D, Medici V, Guerra M, Di Pietro C, Rizzo FR, Musella A, Carola V, Centonze D, Blencowe BJ, Marazziti D, Sette C

Abstract
Tight coordination of gene expression in the developing cerebellum is crucial for establishment of neuronal circuits governing motor and cognitive function. However, transcriptional changes alone do not explain all of the switches underlying neuronal differentiation. Here we unveiled a widespread and highly dynamic splicing program that affects synaptic genes in cerebellar neurons. The motifs enriched in modulated exons implicated the splicing factor Sam68 as a regulator of this program. Sam68 controls splicing of exons with weak branchpoints by directly binding near the 3' splice site and competing with U2AF recruitment. Ablation of Sam68 disrupts splicing regulation of synaptic genes associated with neurodevelopmental diseases and impairs synaptic connections and firing of Purkinje cells, resulting in motor coordination defects, ataxia, and abnormal social behavior. These findings uncover an unexpectedly dynamic splicing regulatory network that shapes the synapse in early life and establishes motor and cognitive circuitry in the developing cerebellum.

PMID: 32492419 [PubMed - as supplied by publisher]



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Three-dimensional niche stiffness synergizes with Wnt7a to modulate the extent of satellite cell symmetric self-renewal divisions.

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Three-dimensional niche stiffness synergizes with Wnt7a to modulate the extent of satellite cell symmetric self-renewal divisions.

Mol Biol Cell. 2020 Jun 03;:mbcE20010078

Authors: Moyle LA, Cheng RY, Liu H, Davoudi S, Ferreira SA, Nissar AA, Sun Y, Gentleman E, Simmons CA, Gilbert PM

Abstract
Satellite cells (SCs), the resident adult stem cells of skeletal muscle, are required for tissue repair throughout life. While many signaling pathways are known to control SC self-renewal, less is known about the mechanisms underlying the spatiotemporal control of self-renewal during skeletal muscle repair. Here, we measured biomechanical changes that accompany skeletal muscle regeneration and determined the implications on SC fate. Using atomic force microscopy, we quantified a 2.9-fold stiffening of the SC niche at time-points associated with planar-oriented symmetric self-renewal divisions. Immunohistochemical analysis confirms increased extracellular matrix deposition within the basal lamina. To test whether three-dimensional (3D) niche stiffness can alter SC behavior or fate, we embedded isolated SC-associated muscle fibers within biochemically inert agarose gels tuned to mimic native tissue stiffness. Time-lapse microscopy revealed that a stiff 3D niche significantly increased the proportion of planar-oriented divisions, without effecting SC viability, fibronectin deposition, or fate change. We then found that 3D niche stiffness synergizes with WNT7a, a biomolecule shown to control SC symmetric self-renewal divisions via the non-canonical WNT/planar cell polarity pathway, to modify stem cell pool expansion. Our results provide new insights into the role of 3D niche biomechanics in regulating SC fate choice. [Media: see text] [Media: see text].

PMID: 32491970 [PubMed - as supplied by publisher]



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Mistranslating tRNA identifies a deleterious S213P mutation in the Saccharomyces cerevisiae eco1-1 allele.

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Mistranslating tRNA identifies a deleterious S213P mutation in the Saccharomyces cerevisiae eco1-1 allele.

Biochem Cell Biol. 2020 May 30;:

Authors: Zhu Y, Berg MD, Yang P, Loll-Krippleber R, Brown GW, Brandl CJ

Abstract
Mistranslation occurs when an amino acid not specified by the standard genetic code is incorporated during translation. Since the ribosome does not read the amino acid, tRNA variants aminoacylated with a non-cognate amino acid or containing a non-cognate anticodon dramatically increase the frequency of mistranslation. In a systematic genetic analysis, we identified a suppression interaction between tRNASerUGG, G26A, which mistranslates proline codons by inserting serine, and eco1-1, a temperature sensitive allele of the gene encoding an acetyltransferase required for sister chromatid cohesion. The suppression was partial with a tRNA that inserts alanine at proline codons and not apparent for a tRNA that inserts serine at arginine codons. Sequencing of the eco1-1 allele revealed a mutation that would convert the highly conserved serine 213 within β7 of the GCN5-related N-acetyltransferase core to proline. Mutation of P213 in eco1-1 back to the wild-type serine restored function of the enzyme at elevated temperature. Our results indicate the utility of mistranslating tRNA variants to identify functionally relevant mutations and identify eco1 as a reporter for mistranslation. We propose that mistranslation could be used as a tool to treat genetic disease.

PMID: 32476470 [PubMed - as supplied by publisher]



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The Rational Development of CD133-Targeting Immunotherapies for Glioblastoma.

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The Rational Development of CD133-Targeting Immunotherapies for Glioblastoma.

Cell Stem Cell. 2020 May 22;:

Authors: Vora P, Venugopal C, Salim SK, Tatari N, Bakhshinyan D, Singh M, Seyfrid M, Upreti D, Rentas S, Wong N, Williams R, Qazi MA, Chokshi C, Ding A, Subapanditha M, Savage N, Mahendram S, Ford E, Adile AA, McKenna D, McFarlane N, Huynh V, Wylie RG, Pan J, Bramson J, Hope K, Moffat J, Singh S

Abstract
CD133 marks self-renewing cancer stem cells (CSCs) in a variety of solid tumors, and CD133+ tumor-initiating cells are known markers of chemo- and radio-resistance in multiple aggressive cancers, including glioblastoma (GBM), that may drive intra-tumoral heterogeneity. Here, we report three immunotherapeutic modalities based on a human anti-CD133 antibody fragment that targets a unique epitope present in glycosylated and non-glycosylated CD133 and studied their effects on targeting CD133+ cells in patient-derived models of GBM. We generated an immunoglobulin G (IgG) (RW03-IgG), a dual-antigen T cell engager (DATE), and a CD133-specific chimeric antigen receptor T cell (CAR-T): CART133. All three showed activity against patient-derived CD133+ GBM cells, and CART133 cells demonstrated superior efficacy in patient-derived GBM xenograft models without causing adverse effects on normal CD133+ hematopoietic stem cells in humanized CD34+ mice. Thus, CART133 cells may be a therapeutically tractable strategy to target CD133+ CSCs in human GBM or other treatment-resistant primary cancers.

PMID: 32464096 [PubMed - as supplied by publisher]



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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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