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Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device.

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Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device.

Acta Crystallogr D Struct Biol. 2019 Dec 01;75(Pt 12):1063-1070

Authors: Rubinstein JL, Guo H, Ripstein ZA, Haydaroglu A, Au A, Yip CM, Di Trani JM, Benlekbir S, Kwok T

Abstract
Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM specimen-preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.

PMID: 31793900 [PubMed - in process]



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Lineage tracing reveals the hierarchical relationship between neural stem cell populations in the mouse forebrain.

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Lineage tracing reveals the hierarchical relationship between neural stem cell populations in the mouse forebrain.

Sci Rep. 2019 Nov 27;9(1):17730

Authors: Sachewsky N, Xu W, Fuehrmann T, van der Kooy D, Morshead CM

Abstract
Since the original isolation of neural stem cells (NSCs) in the adult mammalian brain, further work has revealed a heterogeneity in the NSC pool. Our previous work characterized a distinct, Oct4 expressing, NSC population in the periventricular region, through development and into adulthood. We hypothesized that this population is upstream in lineage to the more abundant, well documented, GFAP expressing NSC. Herein, we show that Oct4 expressing NSCs give rise to neurons, astrocytes and oligodendrocytes throughout the developing brain. Further, transgenic inducible mouse models demonstrate that the rare Oct4 expressing NSCs undergo asymmetric divisions to give rise to GFAP expressing NSCs in naïve and injured brains. This lineage relationship between distinct NSC pools contributes significantly to an understanding of neural development, the NSC lineage in vivo and has implications for neural repair.

PMID: 31776378 [PubMed - in process]



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β2* nAChRs on VTA dopamine and GABA neurons separately mediate nicotine aversion and reward.

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β2* nAChRs on VTA dopamine and GABA neurons separately mediate nicotine aversion and reward.

Proc Natl Acad Sci U S A. 2019 Nov 27;:

Authors: Grieder TE, Besson M, Maal-Bared G, Pons S, Maskos U, van der Kooy D

Abstract
Evidence shows that the neurotransmitter dopamine mediates the rewarding effects of nicotine and other drugs of abuse, while nondopaminergic neural substrates mediate the negative motivational effects. β2* nicotinic acetylcholine receptors (nAChR) are necessary and sufficient for the experience of both nicotine reward and aversion in an intra-VTA (ventral tegmental area) self-administration paradigm. We selectively reexpressed β2* nAChRs in VTA dopamine or VTA γ-amino-butyric acid (GABA) neurons in β2-/- mice to double-dissociate the aversive and rewarding conditioned responses to nicotine in nondependent mice, revealing that β2* nAChRs on VTA dopamine neurons mediate nicotine's conditioned aversive effects, while β2* nAChRs on VTA GABA neurons mediate the conditioned rewarding effects in place-conditioning paradigms. These results stand in contrast to a purely dopaminergic reward theory, leading to a better understanding of the neurobiology of nicotine motivation and possibly to improved therapeutic treatments for smoking cessation.

PMID: 31776253 [PubMed - as supplied by publisher]



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Charge-Balanced Electrical Stimulation Can Modulate Neural Precursor Cell Migration in the Presence of Endogenous Electric Fields in Mouse Brains.

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Charge-Balanced Electrical Stimulation Can Modulate Neural Precursor Cell Migration in the Presence of Endogenous Electric Fields in Mouse Brains.

eNeuro. 2019 Nov 26;:

Authors: Iwasa SN, Rashidi A, Sefton E, Liu NX, Popovic MR, Morshead CM

Abstract
Electric fields can direct cell migration and are crucial during development and tissue repair. We previously reported neural precursor cells are electrosensitive cells that can undergo rapid and directed migration towards the cathode using charge-balanced electrical stimulation in vitro Here, we investigate the ability of electrical stimulation to direct neural precursor migration in mouse brains in vivo To visualize migration, fluorescent adult murine neural precursors were transplanted onto the corpus callosum of adult male mice and intracortical platinum wire electrodes were implanted medial (cathode) and lateral (anode) to the injection site. We applied a charge-balanced biphasic monopolar stimulation waveform for 3 sessions per day, for 3 or 6 days. Irrespective of stimulation, the transplanted neural precursors had a propensity to migrate laterally along the corpus callosum, and applied stimulation affected that migration. Further investigation revealed an endogenous electric field along the corpus callosum that correlated with the lateral migration, suggesting that the applied electric field would need to overcome endogenous cues. There was no difference in transplanted cell differentiation and proliferation, or inflammatory cell numbers near the electrode leads and injection site comparing stimulated and implanted non-stimulated brains. Our results support that endogenous and applied electric fields are important considerations for designing cell therapies for tissue repair in vivo. SIGNIFICANCE STATEMENT The study of electricity in biological environments outside of the well-known action potential is becoming more prominent. Applied electrical stimulation is used clinically and can modulate cell behaviour. Endogenous electric fields exist in the adult brain along the rostral migratory stream and disrupting them can reverse the migration direction of neural precursor cells. We demonstrate that an endogenous electric field exists on the corpus callosum which correlates with the preferred lateral migration of transplanted neural precursor cells. Endogenous electric fields in the brain provide migratory cues that can impact neural repair.

PMID: 31772032 [PubMed - as supplied by publisher]



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Highlights for the 60th anniversary of BBRC.

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Highlights for the 60th anniversary of BBRC.

Biochem Biophys Res Commun. 2019 Dec 17;520(4):699-700

Authors: Stagljar I

PMID: 31761084 [PubMed - in process]



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In-cell identification and measurement of RNA-protein interactions.

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In-cell identification and measurement of RNA-protein interactions.

Nat Commun. 2019 Nov 22;10(1):5317

Authors: Graindorge A, Pinheiro I, Nawrocka A, Mallory AC, Tsvetkov P, Gil N, Carolis C, Buchholz F, Ulitsky I, Heard E, Taipale M, Shkumatava A

Abstract
Regulatory RNAs exert their cellular functions through RNA-binding proteins (RBPs). Identifying RNA-protein interactions is therefore key for a molecular understanding of regulatory RNAs. To date, RNA-bound proteins have been identified primarily through RNA purification followed by mass spectrometry. Here, we develop incPRINT (in cell protein-RNA interaction), a high-throughput method to identify in-cell RNA-protein interactions revealed by quantifiable luminescence. Applying incPRINT to long noncoding RNAs (lncRNAs), we identify RBPs specifically interacting with the lncRNA Firre and three functionally distinct regions of the lncRNA Xist. incPRINT confirms previously known lncRNA-protein interactions and identifies additional interactions that had evaded detection with other approaches. Importantly, the majority of the incPRINT-defined interactions are specific to individual functional regions of the large Xist transcript. Thus, we present an RNA-centric method that enables reliable identification of RNA-region-specific RBPs and is applicable to any RNA of interest.

PMID: 31757954 [PubMed - in process]



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Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing.

Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing.

Cell Rep. 2019 Nov 12;29(7):1739-1746.e5

Authors: Garcia B, Lee J, Edraki A, Hidalgo-Reyes Y, Erwood S, Mir A, Trost CN, Seroussi U, Stanley SY, Cohn RD, Claycomb JM, Sontheimer EJ, Maxwell KL, Davidson AR

Abstract
CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory activity is generally restricted to specific Cas9 homologs. Since Cas9 homologs vary in important properties, differing Cas9s may be optimal for particular genome-editing applications. To facilitate the practical exploitation of multiple Cas9 homologs, here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing. We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction.

PMID: 31722192 [PubMed - in process]



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Cranial irradiation in juvenile mice leads to early and sustained defects in the stem and progenitor cell pools and late cognitive impairments.

Cranial irradiation in juvenile mice leads to early and sustained defects in the stem and progenitor cell pools and late cognitive impairments.

Brain Res. 2019 Nov 09;:146548

Authors: Ruddy RM, Derkach D, Dadwal P, Morshead CM

Abstract
Cranial irradiation is used in combination with other therapies as a treatment for brain tumours and is thought to contribute to long-term cognitive deficits. Several rodent models have demonstrated that these cognitive deficits may be correlated with damage to neural progenitor cells in the subventricular zone (SVZ) and dentate gyrus (DG), the two neurogenic niches of the brain. Studies in rodent models typically assess the proliferating progenitor population, but rarely investigate the effect of cranial irradiation on the neural stem cell pool. Further, few studies evaluate the effects in juveniles, an age when children typically receive this treatment. Herein, we examine the cellular and behavioural effects of juvenile cranial irradiation on stem and progenitor populations in the two neurogenic regions of the brain and assess cognitive outcomes. We found regionally distinct effects of cranial irradiation in the juvenile brain. In the SVZ, we observed a defect in the stem cell pool and a concomitant decrease in proliferating cells that were maintained for at least one week. In the DG, a similar defect in the stem cell pool and proliferating cells was observed and persisted in the stem cell population. Finally, we demonstrated that cranial irradiation resulted in late cognitive deficits. This study demonstrates that juvenile cranial irradiation leads to regionally distinct defects in the stem and progenitor populations, and late cognitive deficits, which may be important factors in determining therapeutic targets and timing of interventions following cranial irradiation.

PMID: 31715143 [PubMed - as supplied by publisher]



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A CD133-AKT-Wnt signaling axis drives glioblastoma brain tumor-initiating cells.

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A CD133-AKT-Wnt signaling axis drives glioblastoma brain tumor-initiating cells.

Oncogene. 2019 Nov 06;:

Authors: Manoranjan B, Chokshi C, Venugopal C, Subapanditha M, Savage N, Tatari N, Provias JP, Murty NK, Moffat J, Doble BW, Singh SK

Abstract
Mechanistic insight into signaling pathways downstream of surface receptors has been revolutionized with integrated cancer genomics. This has fostered current treatment modalities, namely immunotherapy, to capitalize on targeting key oncogenic signaling nodes downstream of a limited number of surface markers. Unfortunately, rudimentary mechanistic understanding of most other cell surface proteins has reduced the clinical utility of these markers. CD133 has reproducibly been shown to correlate with disease progression, recurrence, and poor overall survivorship in the malignant adult brain tumor, glioblastoma (GBM). Using several patient-derived CD133high and CD133low GBMs we describe intrinsic differences in determinants of stemness, which we owe to a CD133-AKT-Wnt signaling axis in which CD133 functions as a putative cell surface receptor for AKT-dependent Wnt activation. These findings may have implications for personalized oncology trials targeting PI3K/AKT or Wnt as both pathways may be activated independent of their canonical drivers, leading to treatment resistance and disease relapse.

PMID: 31695152 [PubMed - as supplied by publisher]



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A potent anti-SpuE antibody allosterically inhibits type III secretion system and attenuates virulence of Pseudomonas aeruginosa.

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A potent anti-SpuE antibody allosterically inhibits type III secretion system and attenuates virulence of Pseudomonas aeruginosa.

J Mol Biol. 2019 Nov 01;:

Authors: Zhang Y, Sun X, Qian Y, Yi H, Song K, Zhu H, Zonta F, Chen W, Ji Q, Miersch S, Sidhu SS, Wu D

Abstract
Multidrug-resistant Gram-negative bacteria infection is particularly severe within the designated ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), which underscores the urgent need to explore alternative therapeutic strategies. The type III secretion system (T3SS) is considered to be a key virulence factor in many Gram-negative bacteria, and T3SS is in turn regulated by SpuE in P. aeruginosa, which is a spermidine binding protein from an ATP-binding cassette transporter family and highly conserved within ESKAPE pathogens. Here, we identified a potent anti-SpuE antagonistic antibody that allosterically inhibits the expression of T3SS and attenuates virulence of P. aeruginosa. X-ray crystallography and molecular dynamics simulations revealed that binding of antibody to SpuE induces a change in the dynamics of SpuE, which in turn may reduce spermidine uptake by P. aeruginosa. The antibody could serve as a template for developing novel biologics to target a broad spectrum of Gram-negative bacteria.

PMID: 31682834 [PubMed - as supplied by publisher]



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