Today's date:

Brad's Most Recent Paper of the Day
Category:   Wnt Signaling Development Childhood genetic diseases Transcription

Enhancing WNT Signaling Restores Cortical Neuronal Spine Maturation and Synaptogenesis in Tbr1 Mutants

Journal: Cell Reports  Publication year: 2020

This is an article I found to be very intriguing, as one of my colleagues when I was at McMaster University, Dr. Karun Singh (soon relocating to the Krembil Brain Insitute in Toronto), studies Autism Spectrum Disorder (ASD), and I had the pleasure of discussing his research with him and his trainees on many occasions. TBR1 (T-box brain transcription factor 1) is a T-box transcription factor in the same family as Brachyury (the original T-box factor), which my lab knows well as a Wnt/β-catenin target gene in embryonic stem cells. Whereas Brachyury regulates mesodermal differentiation, TBR1 plays a critical role in brain development, both pre- and postnatally and is a high-confidence ASD risk gene. The authors had previously shown that TBR1 is required for maintaining corticothalamic identity and synaptogenesis through their studies with conditional knockout mice targeting TBR1 during neonatal development. Here, Fazel Darbandi et al., by employing the same neonatal-specific conditional knockout mouse, report that TBR1 regulates transcriptional circuits in specific cortical brain layers to promote dendritic spine and synaptic density. Of interest to a Wnt signaling fan like me, Wnt signaling is required for proper TBR1 neurodevelopmental function, as Wnt7b is a target gene of TBR1 in a neonatal neurodevelopmental context. The authors show that augmenting Wnt/β-catenin signaling by employing the GSK-3 inhibitors LiCl or SB216763 can ameliorate the effects of TBR1 knockout on dendritic spine and synaptic density. As an aside, please note that SB216763 is not a GSK-3β-specific inhibitor, it will also inhibit GSK-3ɑ.

Category:   Molecular Biology Cell Biology Transcription Viruses

Bad News Wrapped in Protein: Inside the Coronavirus Genome

Journal: New York Times   Year: 2020

This is different from prior "papers of the day" in that it is from a newspaper instead of a science journal. It provides a very nice description of the various components that comprise the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (Covid-19). The author of this article employs analogies, diagrams and clear writing to describe the nucleic acid and protein constituents of SARS-CoV-2 for a lay reader with a basic science background. One does not have to be a virologist or molecular biologist to understand the content of this article, and I highly recommend it for anyone who is interested in learning more about what is currently known (and unknown) about SARS-CoV-2.

Category:   Cool Techniques Genome Editing Molecular Biology Cell Biology Transcription

Robustness of Catalytically-Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells

Journal: Molecular Therapy Nucleic Acids  Publication year: 2020

The original application of the CRISPR/Cas9 system was to generate precisely targeted double-strand breaks that could be repaired by non-homologous end joining or by homology-directed repair when a suitable repair template was provided. Numerous tweaks to this initial system have led to a wide variety of additional applications. The focus of this paper is on the use of catalytically dead Cas9 (dCas9)  fused to transcriptional activators, which can be recruited to specific genomic locations by single-guide RNAs to activate locus-specific transcription. The authors compare first-generation dCas9-VP64 and second-generation dCas9-SAM and dCas9-SunTag to induce gene expression in human pluripotent stem cells and human mesenchymal stem cells. The authors find that all systems induced specific and potent gene expression, but the 2nd-generation systems yielded higher and more consistent increases in expression of target genes.

Category:   Cool Techniques Epigenetics Transcription Cell Biology

ATAC-Me Captures Prolonged DNA Methylation of Dynamic Chromatin Accessibility Loci during Cell Fate Transitions

Journal: Molecular Cell      Publication year: 2020

This is another new technique for investigating chromatin accessibility by using an ATAC-based approach. The cool new alteration to the technique is the incorporation of DNA methylation analysis, which allows for the analysis of chromatin accessibility and DNA methylation from single library preparations. Intriguingly, this technique revealed a disconnect between chromatin accessibility, methylation status and gene activity when it was used to investigate chromatin and transcriptional changes occurring during the differentiation of monocytes to macrophages over a defined time course.

Category:   Cool Techniques Molecular Biology Childhood genetic diseases Cell Biology

Extracellular nanovesicles for packaging of CRISPRCas9 protein and sgRNA to induce therapeutic exon skipping

Journal: Nature Communications    Publication year: 2020

In this paper, the authors describe an elegant system for packaging CRISPRCas9 protein and sgRNAs into extracellular nanovesicles. The extracellular nanovesicle system is termed NanoMEDIC (nanomembrane-derived extracellular vesicles for the delivery of macromolecular cargo). The system employs chemically induced dimerization of FKBP12 and FRB(T2098L) domains with the rapamycin analogue AP21967 to recruit FRB-SpCas9 to an FKBP12-membrane anchoring protein. HIV Tat and Psi+ are used to actively package  ribozyme-sgRNA-ribozyme sequences into the nanovesicles. The authors show that their system is capable of efficiently editing hard-to-transfect cells such as neurons and myoblasts and can successfully induce permanent genomic exon skipping in mouse models (including a the mdx model of muscular dystrophy), which suggests that this system may be of great utility for the therapeutic delivery of genome editing machinery to cells and tissues.

Category:   Cell Biology Development Cancer

Of Cell Shapes and Motion: The Physical Basis of Animal Cell Migration

Journal:  Developmental Cell  Publication year: 2020

I have been working with cells in culture for many years and am used to viewing them under a light microscope, where cells like fibroblasts or embryonic stem cells appear to be more-or-less stationary over minutes of viewing. When the same cells are viewed over a time-course by using time-lapse imaging, it becomes very clear that individual cells within a population are anything but stationary! The processes by which cells move about on cell culture plates, extracellular matrix, or other surfaces are beginning to be understood at increasingly detailed levels. In this review, the authors provide a comprehensive overview of the mechanisms employed by metazoan cells to confer motility. The authors discuss the implications of different modes of cellular migration in normal development and in pathological events such as cancer cell metastasis.

Category:   Cancer Transcription Post-transcriptional Regulation Epigenetics Cell Biology

m6A Modification in Coding and Non-coding RNAs: Roles and Therapeutic Implications in Cancer

Journal:  Cancer Cell   Publication year: 2020

N6-Methyladenosine (m6A) RNA modification is a reversible modification that occurs on mRNA and lncRNA transcripts. This review describes the enzymes responsible for methylating and demethylating m6A, "readers" of m6A-marked RNA, and the impact m6A modification can have on transcript function. For those studying transcriptional and post-transcriptional regulation this review serves as an excellent resource to help one catch up on what is known about m6A modification of coding and non-coding RNAs.

Category:   Cool Techniques Molecular Biology Genome Editing

Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures

Journal: Cell Reports   Publication year: 2020

This is a "cool techniques" paper that provides an easy and quick way with which to detect precision genome editing events. The authors' DTECT (Dinucleotide signaTurE CapTure) technique uses the type IIS restriction enzyme AcuI as a tool with which to evaluate target sequences in their wild-type and genome-edited states. This is a great addition to the toolbox of those employing precision genome editing in their research.

Category:   Stem Cells Regenerative Medicine Wnt Signaling Development Proteomics Cell Biology

Proteomic profiling of stem cell tissues during regeneration of deer antler - a model of mammalian organ regeneration

Journal: Journal of Proteome Research Publication year: 2020

I was only made aware of the regenerative properties of deer antler late last year at the Till & McCulloch Stem Cell meeting in Montreal. Deer antlers regenerate themselves annually and studying the details of this process could provide insights to guide new regenerative medicine strategies, especially for tissues generated from neural crest-derived stem cells. In this paper, the authors use a label-free proteomic approach to compare the protein expression profiles in different stem cell tissues of the antler. The datasets the authors generated are readily available and will be a useful resource for those studying regeneration. For those interested in Wnt and Hippo signaling, these pathways featured prominently in the data obtained from "activated" antler stem cells.

Category:   Stem Cells Cell Biology Wnt Signaling Transcription

Ascl2-Dependent Cell Dedifferentiation Drives Regeneration of Ablated Intestinal Stem Cells

Journal: Cell Stem Cell    Publication year: 2020

In this paper, the authors use lineage tracing and selective ablation of Lgr5(+) intestinal stem cells to show that intestinal stem cell regeneration occurs through dedifferentiation of recently spawned stem cell descendants. This finding challenges the previously held view that +4 reserve stem cells were responsible for replacing ablated Lgr5(+) stem cells of the intestinal crypt. The authors highlight the importance of the transcription factor Ascl2 in mediating the dedifferentiation required to give rise to nascent replacement stem cells.

Category:   Cancer Stem Cells Wnt Signaling Cell Biology Cool Techniques

Plasticity of Lgr5-Negative Cancer Cells Drives Metastasis in Colorectal Cancer

Journal:  Cell Stem Cell   Publication year: 2020

This is another cool paper that is highly relevant to those studying cancer stem cell (CSC) biology, especially in cases where Lgr5-positive CSCs are involved. The authors examine the progression of colorectal cancer (CRC) in a mouse model comprising tamoxifen-inducible Villin-driven Cre, LoxP-flanked APC, KRAS-(LSL-G12D), TP53(KO/KO), Lgr5(DTR-eGFP), and R26R-confetti, which allows for the visualization of LGR5-positive cancer stem cells and selective ablation of these CSCs by using diphtheria toxin. By using their elegant system, the authors find that the tumour cells that migrate, disseminate, and colonize metastases are mainly Lgr5-negative, and Lgr5-negative metastases have the ability to develop an epithelial hierarchy, due to their intrinsic phenotypic plasticity. The authors also use human-mouse xenografts to substantiate the relevance of their mouse model findings to human CRC. This paper has important implications for treatment strategies focused on CSCs and CSC-maintaining niche factors, which do not generally consider endogenous cellular plasticity during the metastatic program.

Category:   Cool Techniques Cell Biology Molecular Biology Post-transcriptional regulation Transcription

The biogenesis, biology and characterization of circular RNAs

Journal: Nature Reviews Genetics     Publication year: 2019

Based on some recent discoveries in the lab, I have become very interested in non-coding RNAs, especially long-non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). This is a recent review on circular RNAs that provides a nice overview of our current understanding of the biology of circular RNAs and the ways in which circular RNAs are studied. Circular RNAs are important regulators of cellular function and are known to be dysregulated in several human diseases. Their circular nature makes them somewhat difficult to study, but new bioinformatic tools specifically designed for circRNA characterization and quantitation are helping researchers investigate these molecules. I highly recommend this review for those unfamiliar with circRNAs.

The Doble Lab

Basic Medical Sciences Building
University of Manitoba
Room 615-744 Bannatyne Avenue
Winnipeg, Manitoba
Canada, R3E 0W2