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In vitro Evaluation of ASCs and HUVECs Co-cultures in 3D Biodegradable Hydrogels on Neurite Outgrowth and Vascular Organization.

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In vitro Evaluation of ASCs and HUVECs Co-cultures in 3D Biodegradable Hydrogels on Neurite Outgrowth and Vascular Organization.

Front Cell Dev Biol. 2020;8:489

Authors: Rocha LA, Gomes ED, Afonso JL, Granja S, Baltazar F, Silva NA, Shoichet MS, Sousa RA, Learmonth DA, Salgado AJ

Abstract
Vascular disruption following spinal cord injury (SCI) decisively contributes to the poor functional recovery prognosis facing patients with the condition. Using a previously developed gellan gum hydrogel to which the adhesion motif GRGDS was grafted (GG-GRGDS), this work aimed to understand the ability of adipose-derived stem cells (ASCs) to impact vascular organization of human umbilical vein endothelial cells (HUVECs), and how this in turn affects neurite outgrowth of dorsal root ganglia (DRG) explants. Our data shows that culturing these cells together lead to a synergistic effect as showed by increased stimulation of neuritogenesis on DRG. Importantly, HUVECs were only able to assemble into vascular-like structures when cultured in the presence of ASCs, which shows the capacity of these cells in reorganizing the vascular milieu. Analysis of selected neuroregulatory molecules showed that the co-culture upregulated the secretion of several neurotrophic factors. On the other hand, ASCs, and ASCs + HUVECs presented a similar profile regarding the presence of angiotrophic molecules herein analyzed. Finally, the implantation of GG-GRGDS hydrogels encapsulating ASCs in the chick chorioallantoic membrane (CAM) lead to increases in vascular recruitment toward the hydrogels in comparison to GG-GRGDS alone. This indicates that the combination of ASCs with GG-GRGDS hydrogels could promote re-vascularization in trauma-related injuries in the central nervous system and thus control disease progression and induce functional recovery.

PMID: 32612997 [PubMed]



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Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

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Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

Cell. 2020 Jun 23;:

Authors: Lee H, Beilhartz GL, Kucharska I, Raman S, Cui H, Lam MHY, Liang H, Rubinstein JL, Schramek D, Julien JP, Melnyk RA, Taipale M

Abstract
Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.

PMID: 32589945 [PubMed - as supplied by publisher]



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Exploring whole-genome duplicate gene retention with complex genetic interaction analysis.

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Exploring whole-genome duplicate gene retention with complex genetic interaction analysis.

Science. 2020 Jun 26;368(6498):

Authors: Kuzmin E, VanderSluis B, Nguyen Ba AN, Wang W, Koch EN, Usaj M, Khmelinskii A, Usaj MM, van Leeuwen J, Kraus O, Tresenrider A, Pryszlak M, Hu MC, Varriano B, Costanzo M, Knop M, Moses A, Myers CL, Andrews BJ, Boone C

Abstract
Whole-genome duplication has played a central role in the genome evolution of many organisms, including the human genome. Most duplicated genes are eliminated, and factors that influence the retention of persisting duplicates remain poorly understood. We describe a systematic complex genetic interaction analysis with yeast paralogs derived from the whole-genome duplication event. Mapping of digenic interactions for a deletion mutant of each paralog, and of trigenic interactions for the double mutant, provides insight into their roles and a quantitative measure of their functional redundancy. Trigenic interaction analysis distinguishes two classes of paralogs: a more functionally divergent subset and another that retained more functional overlap. Gene feature analysis and modeling suggest that evolutionary trajectories of duplicated genes are dictated by combined functional and structural entanglement factors.

PMID: 32586993 [PubMed - in process]



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Functional genomics identifies new synergistic therapies for retinoblastoma.

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Functional genomics identifies new synergistic therapies for retinoblastoma.

Oncogene. 2020 Jun 22;:

Authors: Aubry A, Pearson JD, Huang K, Livne-Bar I, Ahmad M, Jagadeesan M, Khetan V, Ketela T, Brown KR, Yu T, Lu S, Wrana JL, Moffat J, Bremner R

Abstract
Local intravitreal or intra-arterial chemotherapy has improved therapeutic success for the pediatric cancer retinoblastoma (RB), but toxicity remains a major caveat. RB initiates primarily with RB1 loss or, rarely, MYCN amplification, but the critical downstream networks are incompletely understood. We set out to uncover perturbed molecular hubs, identify synergistic drug combinations to target these vulnerabilities, and expose and overcome drug resistance. We applied dynamic transcriptomic analysis to identify network hubs perturbed in RB versus normal fetal retina, and performed in vivo RNAi screens in RB1null and RB1wt;MYCNamp orthotopic xenografts to pinpoint essential hubs. We employed in vitro and in vivo studies to validate hits, define mechanism, develop new therapeutic modalities, and understand drug resistance. We identified BRCA1 and RAD51 as essential for RB cell survival. Their oncogenic activity was independent of BRCA1 functions in centrosome, heterochromatin, or ROS regulation, and instead linked to DNA repair. RAD51 depletion or inhibition with the small molecule inhibitor, B02, killed RB cells in a Chk1/Chk2/p53-dependent manner. B02 further synergized with clinically relevant topotecan (TPT) to engage this pathway, activating p53-BAX mediated killing of RB but not human retinal progenitor cells. Paradoxically, a B02/TPT-resistant tumor exhibited more DNA damage than sensitive RB cells. Resistance reflected dominance of the p53-p21 axis, which mediated cell cycle arrest instead of death. Deleting p21 or applying the BCL2/BCL2L1 inhibitor Navitoclax re-engaged the p53-BAX axis, and synergized with B02, TPT or both to override resistance. These data expose new synergistic therapies to trigger p53-induced killing in diverse RB subtypes.

PMID: 32572160 [PubMed - as supplied by publisher]



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Microexons: at the nexus of nervous system development, behaviour and autism spectrum disorder.

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Microexons: at the nexus of nervous system development, behaviour and autism spectrum disorder.

Curr Opin Genet Dev. 2020 Jun 11;65:22-33

Authors: Gonatopoulos-Pournatzis T, Blencowe BJ

Abstract
The discovery and characterization of a network of highly conserved neuronal microexons have provided fundamental new insight into mechanisms underlying nervous system development and function, as well as an important basis for pathway convergence in autism spectrum disorder. In the past few years, considerable progress has been made in comprehensively determining the repertoires of factors that control neuronal microexons. These results have illuminated molecular mechanisms that activate the splicing of microexons, including those that control gene expression programs critical for neurogenesis, as well as synaptic protein translation and neuronal activity. Remarkably, individual disruption of specific microexons in these pathways results in autism-like phenotypes and cognitive impairment in mice. This review discusses these findings and their implications for delivering new therapeutic strategies for neurological disorders.

PMID: 32535349 [PubMed - as supplied by publisher]



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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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