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Targeting ALK2: An Open Science Approach to Developing Therapeutics for the Treatment of Diffuse Intrinsic Pontine Glioma.

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Targeting ALK2: An Open Science Approach to Developing Therapeutics for the Treatment of Diffuse Intrinsic Pontine Glioma.

J Med Chem. 2020 May 05;:

Authors: Ensan D, Smil D, Zepeda-Velázquez CA, Panagopoulos D, Wong JF, Williams EP, Adamson R, Bullock AN, Kiyota T, Aman A, Roberts OG, Edwards AM, O'Meara JA, Isaac MB, Al-Awar R

Abstract
Diffuse intrinsic pontine glioma is an aggressive pediatric cancer for which no effective chemotherapeutic drugs exist. Analysis of the genomic landscape of this disease has led to the identification of the serine/threonine kinase ALK2 as a potential target for therapeutic intervention. In this work, we adopted an open science approach to develop a series of potent type I inhibitors of ALK2 which are orally bio-available and brain-penetrant. Initial efforts resulted in the discovery of M4K2009, an analogue of the previously reported ALK2 inhibitor LDN-214117. Although highly selective for ALK2 over the TGF-βR1 receptor ALK5, M4K2009 is also moderately active against the hERG potassium channel. Varying the substituents of the trimethoxyphenyl moiety gave rise to an equipotent benzamide analogue M4K2149 with reduced off-target affinity for the ion channel. Additional modifications yielded 2-fluoro-6-methoxybenzamide derivatives (26a-c), which possess high inhibitory activity against ALK2, excellent selectivity, and superior pharmacokinetic profiles.

PMID: 32369358 [PubMed - as supplied by publisher]



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Direct loading of blood for plasma separation and diagnostic assays on a digital microfluidic device.

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Direct loading of blood for plasma separation and diagnostic assays on a digital microfluidic device.

Lab Chip. 2020 Apr 27;:

Authors: Dixon C, Lamanna J, Wheeler AR

Abstract
Finger-stick blood sampling is convenient for point of care diagnostics, but whole blood samples are problematic for many assays because of severe matrix effects associated with blood cells and cell debris. We introduce a new digital microfluidic (DMF) diagnostic platform with integrated porous membranes for blood-plasma separation from finger-stick blood volumes, capable of performing complex, multi-step, diagnostic assays. Importantly, the samples can be directly loaded onto the device by a finger "dab" for user-friendly operation. We characterize the platform by comparison to plasma generated via the "gold standard" centrifugation technique, and demonstrate a 21-step rubella virus (RV) IgG immunoassay yielding a detection limit of 1.9 IU mL-1, below the diagnostic cut-off. We propose that this work represents a critical next step in DMF based portable diagnostic assays-allowing the analysis of whole blood samples without pre-processing.

PMID: 32338260 [PubMed - as supplied by publisher]



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A 96-well culture platform enables longitudinal analyses of engineered human skeletal muscle microtissue strength.

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A 96-well culture platform enables longitudinal analyses of engineered human skeletal muscle microtissue strength.

Sci Rep. 2020 Apr 24;10(1):6918

Authors: Afshar ME, Abraha HY, Bakooshli MA, Davoudi S, Thavandiran N, Tung K, Ahn H, Ginsberg HJ, Zandstra PW, Gilbert PM

Abstract
Three-dimensional (3D) in vitro models of human skeletal muscle mimic aspects of native tissue structure and function, thereby providing a promising system for disease modeling, drug discovery or pre-clinical validation, and toxicity testing. Widespread adoption of this research approach is hindered by the lack of easy-to-use platforms that are simple to fabricate and that yield arrays of human skeletal muscle micro-tissues (hMMTs) in culture with reproducible physiological responses that can be assayed non-invasively. Here, we describe a design and methods to generate a reusable mold to fabricate a 96-well platform, referred to as MyoTACTIC, that enables bulk production of 3D hMMTs. All 96-wells and all well features are cast in a single step from the reusable mold. Non-invasive calcium transient and contractile force measurements are performed on hMMTs directly in MyoTACTIC, and unbiased force analysis occurs by a custom automated algorithm, allowing for longitudinal studies of function. Characterizations of MyoTACTIC and resulting hMMTs confirms the capability of the device to support formation of hMMTs that recapitulate biological responses. We show that hMMT contractile force mirrors expected responses to compounds shown by others to decrease (dexamethasone, cerivastatin) or increase (IGF-1) skeletal muscle strength. Since MyoTACTIC supports hMMT long-term culture, we evaluated direct influences of pancreatic cancer chemotherapeutics agents on contraction competent human skeletal muscle myotubes. A single application of a clinically relevant dose of Irinotecan decreased hMMT contractile force generation, while clear effects on myotube atrophy were observed histologically only at a higher dose. This suggests an off-target effect that may contribute to cancer associated muscle wasting, and highlights the value of the MyoTACTIC platform to non-invasively predict modulators of human skeletal muscle function.

PMID: 32332853 [PubMed - as supplied by publisher]



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Functional arrays of human pluripotent stem cell-derived cardiac microtissues.

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Functional arrays of human pluripotent stem cell-derived cardiac microtissues.

Sci Rep. 2020 Apr 24;10(1):6919

Authors: Thavandiran N, Hale C, Blit P, Sandberg ML, McElvain ME, Gagliardi M, Sun B, Witty A, Graham G, Do VTH, Bakooshli MA, Le H, Ostblom J, McEwen S, Chau E, Prowse A, Fernandes I, Norman A, Gilbert PM, Keller G, Tagari P, Xu H, Radisic M, Zandstra PW

Abstract
To accelerate the cardiac drug discovery pipeline, we set out to develop a platform that would be capable of quantifying tissue-level functions such as contractile force and be amenable to standard multiwell-plate manipulations. We report a 96-well-based array of 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues - termed Cardiac MicroRings (CaMiRi) - in custom 3D-print-molded multiwell plates capable of contractile force measurement. Within each well, two elastomeric microcantilevers are situated above a circumferential ramp. The wells are seeded with cell-laden collagen, which, in response to the gradual slope of the circumferential ramp, self-organizes around tip-gated microcantilevers to form contracting CaMiRi. The contractile force exerted by the CaMiRi is measured and calculated using the deflection of the cantilevers. Platform responses were robust and comparable across wells, and we used it to determine an optimal tissue formulation. We validated the contractile force response of CaMiRi using selected cardiotropic compounds with known effects. Additionally, we developed automated protocols for CaMiRi seeding, image acquisition, and analysis to enable the measurement of contractile force with increased throughput. The unique tissue fabrication properties of the platform, and the consequent effects on tissue function, were demonstrated upon adding hPSC-derived epicardial cells to the system. This platform represents an open-source contractile force screening system useful for drug screening and tissue engineering applications.

PMID: 32332814 [PubMed - as supplied by publisher]



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BraInMap Elucidates the Macromolecular Connectivity Landscape of Mammalian Brain.

BraInMap Elucidates the Macromolecular Connectivity Landscape of Mammalian Brain.

Cell Syst. 2020 Apr 22;10(4):333-350.e14

Authors: Pourhaghighi R, Ash PEA, Phanse S, Goebels F, Hu LZM, Chen S, Zhang Y, Wierbowski SD, Boudeau S, Moutaoufik MT, Malty RH, Malolepsza E, Tsafou K, Nathan A, Cromar G, Guo H, Abdullatif AA, Apicco DJ, Becker LA, Gitler AD, Pulst SM, Youssef A, Hekman R, Havugimana PC, White CA, Blum BC, Ratti A, Bryant CD, Parkinson J, Lage K, Babu M, Yu H, Bader GD, Wolozin B, Emili A

Abstract
Connectivity webs mediate the unique biology of the mammalian brain. Yet, while cell circuit maps are increasingly available, knowledge of their underlying molecular networks remains limited. Here, we applied multi-dimensional biochemical fractionation with mass spectrometry and machine learning to survey endogenous macromolecules across the adult mouse brain. We defined a global "interactome" comprising over one thousand multi-protein complexes. These include hundreds of brain-selective assemblies that have distinct physical and functional attributes, show regional and cell-type specificity, and have links to core neurological processes and disorders. Using reciprocal pull-downs and a transgenic model, we validated a putative 28-member RNA-binding protein complex associated with amyotrophic lateral sclerosis, suggesting a coordinated function in alternative splicing in disease progression. This brain interaction map (BraInMap) resource facilitates mechanistic exploration of the unique molecular machinery driving core cellular processes of the central nervous system. It is publicly available and can be explored here https://www.bu.edu/dbin/cnsb/mousebrain/.

PMID: 32325033 [PubMed - as supplied by publisher]



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Robust production of uniform human cerebral organoids from pluripotent stem cells.

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Robust production of uniform human cerebral organoids from pluripotent stem cells.

Life Sci Alliance. 2020 May;3(5):

Authors: Sivitilli AA, Gosio JT, Ghoshal B, Evstratova A, Trcka D, Ghiasi P, Hernandez JJ, Beaulieu JM, Wrana JL, Attisano L

Abstract
Human cerebral organoid (hCO) models offer the opportunity to understand fundamental processes underlying human-specific cortical development and pathophysiology in an experimentally tractable system. Although diverse methods to generate brain organoids have been developed, a major challenge has been the production of organoids with reproducible cell type heterogeneity and macroscopic morphology. Here, we have directly addressed this problem by establishing a robust production pipeline to generate morphologically consistent hCOs and achieve a success rate of >80%. These hCOs include both a radial glial stem cell compartment and electrophysiologically competent mature neurons. Moreover, we show using immunofluorescence microscopy and single-cell profiling that individual organoids display reproducible cell type compositions that are conserved upon extended culture. We expect that application of this method will provide new insights into brain development and disease processes.

PMID: 32303588 [PubMed - as supplied by publisher]



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Mitochondrial carrier homolog 2 (MTCH2) is necessary for AML survival.

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Mitochondrial carrier homolog 2 (MTCH2) is necessary for AML survival.

Blood. 2020 Apr 16;:

Authors: Khan DH, Mullokandov M, Wu Y, Voisin V, Gronda MV, Hurren R, Wang X, MacLean N, Jeyaraju DV, Jitkova Y, Xu GW, Laister RC, Seneviratne A, Blatman Z, Ketala T, Bader GD, Marhon SA, Carvalho DD, Minden MD, Gross A, Schimmer AD

Abstract
Through a CRISPR screen to identify mitochondrial genes necessary for the growth of AML cells, we identified the mitochondrial outer membrane protein MTCH2 (Mitochondrial Carrier Homolog 2). In AML, knockdown of MTCH2 decreased growth, reduced engraftment potential of stem cells and induced differentiation. Inhibiting MTCH2 in AML cells increased nuclear pyruvate and pyruvate dehydrogenase that induced histone acetylation and subsequently promoted the differentiation of AML cells. Thus, we have defined a new mechanism by which mitochondria and metabolism regulate AML stem cells and gene expression.

PMID: 32299104 [PubMed - as supplied by publisher]



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Performance of soluble Klotho assays in clinical samples of kidney disease.

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Performance of soluble Klotho assays in clinical samples of kidney disease.

Clin Kidney J. 2020 Apr;13(2):235-244

Authors: Neyra JA, Moe OW, Pastor J, Gianella F, Sidhu SS, Sarnak MJ, Ix JH, Drew DA

Abstract
Background: Soluble Klotho has multiple systemic salutary effects. In animals, both acute and chronic kidney disease models display systemic Klotho deficiency. As such, there is considerable interest in investigating soluble Klotho as a biomarker in patients with different types and severity of kidney diseases. Unfortunately, there remains uncertainty regarding the best method to measure soluble Klotho in human serum samples.
Methods: Using human serum samples obtained from several clinical cohorts with a wide range of kidney function, we measured soluble Klotho using a commercial enzyme-linked immunosorbent assay (ELISA) as well as with an immunoprecipitation-immunoblot (IP-IB) assay utilizing a synthetic antibody with high affinity and specificity for Klotho. Recovery of spiking with a known amount of exogenous Klotho was tested. A subset of samples was analyzed with and without the addition of a protease inhibitor cocktail at the time of collection or after the first freeze-thaw cycle to determine if these maneuvers influenced performance.
Results: The IP-IB assay was superior to the ELISA at recovery of exogenous Klotho (81-115% versus 60-81%) across the spectrum of kidney function. Klotho measurements by IP-IB were highly correlated with estimated glomerular filtration rate (eGFR) (R = 0.80, P < 0.001) in comparison with the commercial ELISA, which exhibited minimal correlation with eGFR (R = 0.18, P = 0.12). Use of a protease inhibitor cocktail neither improved nor impaired performance of the IP-IB assay; however, subsequent freeze-thaw cycle resulted in a significant reduction in Klotho recovery and dissipated the correlation between Klotho levels and eGFR. With the ELISA, the use of protease inhibitor cocktail resulted in an increase in intrasubject variability.
Conclusions: The IP-IB assay is preferable to the commercial ELISA to measure soluble Klotho concentrations in never-thawed serum samples of humans with varying severity of kidney disease. However, due to the labor-intensive nature of the IP-IB assay, further research is needed to secure an assay suitable for high-throughput work.

PMID: 32297879 [PubMed - as supplied by publisher]



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A reference map of the human binary protein interactome.

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A reference map of the human binary protein interactome.

Nature. 2020 04;580(7803):402-408

Authors: Luck K, Kim DK, Lambourne L, Spirohn K, Begg BE, Bian W, Brignall R, Cafarelli T, Campos-Laborie FJ, Charloteaux B, Choi D, Coté AG, Daley M, Deimling S, Desbuleux A, Dricot A, Gebbia M, Hardy MF, Kishore N, Knapp JJ, Kovács IA, Lemmens I, Mee MW, Mellor JC, Pollis C, Pons C, Richardson AD, Schlabach S, Teeking B, Yadav A, Babor M, Balcha D, Basha O, Bowman-Colin C, Chin SF, Choi SG, Colabella C, Coppin G, D'Amata C, De Ridder D, De Rouck S, Duran-Frigola M, Ennajdaoui H, Goebels F, Goehring L, Gopal A, Haddad G, Hatchi E, Helmy M, Jacob Y, Kassa Y, Landini S, Li R, van Lieshout N, MacWilliams A, Markey D, Paulson JN, Rangarajan S, Rasla J, Rayhan A, Rolland T, San-Miguel A, Shen Y, Sheykhkarimli D, Sheynkman GM, Simonovsky E, Taşan M, Tejeda A, Tropepe V, Twizere JC, Wang Y, Weatheritt RJ, Weile J, Xia Y, Yang X, Yeger-Lotem E, Zhong Q, Aloy P, Bader GD, De Las Rivas J, Gaudet S, Hao T, Rak J, Tavernier J, Hill DE, Vidal M, Roth FP, Calderwood MA

Abstract
Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships1,2. Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome3, transcriptome4 and proteome5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.

PMID: 32296183 [PubMed - indexed for MEDLINE]



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Medulloblastoma Arises from the Persistence of a Rare and Transient Sox2+ Granule Neuron Precursor.

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Medulloblastoma Arises from the Persistence of a Rare and Transient Sox2+ Granule Neuron Precursor.

Cell Rep. 2020 Apr 14;31(2):107511

Authors: Selvadurai HJ, Luis E, Desai K, Lan X, Vladoiu MC, Whitley O, Galvin C, Vanner RJ, Lee L, Whetstone H, Kushida M, Nowakowski T, Diamandis P, Hawkins C, Bader G, Kriegstein A, Taylor MD, Dirks PB

Abstract
Medulloblastoma (MB) is a neoplasm linked to dysregulated cerebellar development. Previously, we demonstrated that the Sonic Hedgehog (SHH) subgroup grows hierarchically, with Sox2+ cells at the apex of tumor progression and relapse. To test whether this mechanism is rooted in a normal developmental process, we studied the role of Sox2 in cerebellar development. We find that the external germinal layer (EGL) is derived from embryonic Sox2+ precursors and that the EGL maintains a rare fraction of Sox2+ cells during the first postnatal week. Through lineage tracing and single-cell analysis, we demonstrate that these Sox2+ cells are within the Atoh1+ lineage, contribute extensively to adult granule neurons, and resemble Sox2+ tumor cells. Critically, constitutive activation of the SHH pathway leads to their aberrant persistence in the EGL and rapid tumor onset. We propose that failure to eliminate this rare but potent developmental population is the tumor initiation mechanism in SHH-subgroup MB.

PMID: 32294450 [PubMed - as supplied by publisher]



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