Defining the Cis-Regulatory Elements of SCN1A in GABAergic Interneurons.

Dravet syndrome (DS) is a severe neurodevelopmental disorder associated with physical and cognitive impairments, often attributed to mutations in the SCN1A gene. However, gene regulatory features that modulate SCN1A expression found in vivo and in vitro models remain elusive. This study elucidates cell type-specific cis-regulatory elements (CRE) regulating SCN1A expression in cortical fast-spiking GABAergic inhibitory interneurons, the predominantly affected cell type in DS.

A CRISPR/Cas9 screen reveals proteins at the endosome-Golgi interface that modulate cellular anti-sense oligonucleotide activity.

Anti-sense oligonucleotides (ASOs) are modified synthetic single-stranded molecules with enhanced stability, activity, and bioavailability. They associate with RNA through sequence complementarity and can reduce or alter mRNA expression upon binding of splice site positions. To target RNA in the nucleus or cytoplasm, ASOs must cross membranes, a poorly understood process.

An efficient, non-viral arrayed CRISPR screening platform for iPSC-derived myeloid and microglia models.

Here, we developed a CRISPR-Cas9 arrayed screen to investigate lipid handling pathways in human induced pluripotent stem cell (iPSC)-derived microglia. We established a robust method for the nucleofection of CRISPR-Cas9 ribonucleoprotein complexes into iPSC-derived myeloid cells, enabling genetic perturbations. Using this approach, we performed a targeted screen to identify key regulators of lipid droplet formation dependent on Apolipoprotein E (APOE).

RIP1 inhibition protects retinal ganglion cells in glaucoma models of ocular injury.

Receptor-interacting protein 1 (RIP1, RIPK1) is a critical mediator of multiple signaling pathways that promote inflammatory responses and cell death. The kinase activity of RIP1 contributes to the pathogenesis of a number of inflammatory and neurodegenerative diseases. However, the role of RIP1 in retinopathies remains unclear.

A fluorescent splice-switching mouse model enables high-throughput, sensitive quantification of antisense oligonucleotide delivery and activity.

While antisense oligonucleotides (ASOs) are used in the clinic, therapeutic development is hindered by the inability to assay ASO delivery and activity in vivo. Accordingly, we developed a dual-fluorescence, knockin mouse model that constitutively expresses mKate2 and an engineered EGFP that is alternatively spliced in the presence of ASO to induce expression. We first examined free ASO activity in the brain following intracerebroventricular injection revealing EGFP splice-switching is both ASO concentration and time dependent in major central nervous system cell types.

Preventing VEGF-Mediated Vascular Permeability by Experimentally Potentiating BBB Characteristics in Endothelial Cells.

Difficulties with poor reproducibility and translatability of animal model-based research, along with increased efforts to abide by the 3Rs tenet of animal welfare, are driving demand for more relevant human cellular systems. This is especially true for central nervous system (CNS) vasculatures with specialized properties and barriers, namely the blood-brain and blood-retinal barriers (BBB and BRB, respectively) which are difficult to model in vitro. The BBB and BRB protect neurovascular units by regulating nutrient homeostasis, maintaining local ion levels, protecting against exposure from circulating toxins and pathogens, and restricting passage of peripheral immune factors.

Decoding Neuronal Diversification by Multiplexed Single-cell RNA-Seq.

Cellular reprogramming is driven by a defined set of transcription factors; however, the regulatory logic that underlies cell-type specification and diversification remains elusive. Single-cell RNA-seq provides unprecedented coverage to measure dynamic molecular changes at the single-cell resolution. Here, we multiplex and ectopically express 20 pro-neuronal transcription factors in human dermal fibroblasts and demonstrate a widespread diversification of neurons based on cell morphology and canonical neuronal marker expressions.

Inducers of the endothelial cell barrier identified through chemogenomic screening in genome-edited hPSC-endothelial cells.

The blood-retina barrier and blood-brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity.

Characterization of Tumor Blood Vasculature Expression of Human Invasive Bladder Cancer by Laser Capture Microdissection and Transcriptional Profiling.

Tumor-associated blood vessels differ from normal vessels and play key roles in tumor progression. We aimed to identify biomolecules that are expressed differentially in human bladder cancer-associated blood vessels to find novel biomarkers and mechanisms involved in tumor-associated angiogenesis. The transcriptome of tumor blood vasculature from human invasive bladder carcinoma (I-BLCA) and normal bladder tissue vasculature was compared using differential expression and unsupervised hierarchical clustering analyses.

Functional Genomics for Cancer Drug Target Discovery.

Functional genomics describes a field of biology that uses a range of approaches for assessing gene function with high-throughput molecular, genetic, and cellular technologies. The near limitless potential for applying these concepts to study the activities of all genetic loci has completely upended how today's cancer biologists tackle drug target discovery. We provide an overview of contemporary functional genomics platforms, highlighting areas of distinction and complementarity across technologies, so as to aid in the development or interpretation of cancer-focused screening efforts.

Patient hiPSCs Identify Vascular Smooth Muscle Arylacetamide Deacetylase as Protective against Atherosclerosis.

Although susceptibility to cardiovascular disease (CVD) is different for every patient, why some patients with type 2 diabetes mellitus (T2DM) develop CVD while others are protected has not yet been clarified. Using T2DM-patient-derived human induced pluripotent stem cells (hiPSCs), we found that in patients protected from CVD, there was significantly elevated expression of an esterase, arylacetamide deacetylase (AADAC), in vascular smooth muscle cells (VSMCs). We overexpressed this esterase in human primary VSMCs and VSMCs differentiated from hiPSCs and observed that the number of lipid droplets was significantly diminished.

Identification of a combination of transcription factors that synergistically increases endothelial cell barrier resistance.

Endothelial cells (ECs) display remarkable plasticity during development before becoming quiescent and functionally mature. EC maturation is directed by several known transcription factors (TFs), but the specific set of TFs responsible for promoting high-resistance barriers, such as the blood-brain barrier (BBB), have not yet been fully defined. Using expression mRNA data from published studies on ex vivo ECs from the central nervous system (CNS), we predicted TFs that induce high-resistance barrier properties of ECs as in the BBB.

Modeling the Effects of Severe Metabolic Disease by Genome Editing of hPSC-Derived Endothelial Cells Reveals an Inflammatory Phenotype.

The kinase AKT2 (PKB) is an important mediator of insulin signaling, for which loss-of-function knockout (KO) mutants lead to early onset diabetes mellitus, and dominant active mutations lead to early development of obesity and endothelial cell (EC) dysfunction. To model EC dysfunction, we used edited human pluripotent stem cells (hPSCs) that carried either a homozygous deletion of AKT2 (AKT2 KO) or a dominant active mutation (AKT2 E17K), which, along with the parental wild type (WT), were differentiated into ECs. Profiling of EC lines indicated an increase in proinflammatory and a reduction in anti-inflammatory fatty acids, an increase in inflammatory chemokines in cell supernatants, increased expression of proinflammatory genes, and increased binding to the EC monolayer in a functional leukocyte adhesion assay for both AKT2 KO and AKT2 E17K.

Cellular Resistance Mechanisms to Targeted Protein Degradation Converge Toward Impairment of the Engaged Ubiquitin Transfer Pathway.

Proteolysis targeting chimeras are bifunctional small molecules capable of recruiting a target protein of interest to an E3 ubiquitin ligase that facilitates target ubiquitination followed by proteasome-mediated degradation. The first molecules acting on this novel therapeutic paradigm have just entered clinical testing. Here, by using Bromodomain Containing 4 (BRD4) degraders engaging cereblon and Von Hippel-Lindau E3 ligases, we investigated key determinants of resistance to this new mode of action.

Monolayer Generation of Vascular Endothelial Cells from Human Pluripotent Stem Cells.

The use of human pluripotent stem cells (hPSCs) for modeling human diseases and therapeutic applications requires differentiation methods that generate physiologically relevant cell types in a robust and standardized way. Herein, we describe an efficient and scalable monolayer protocol to convert pluripotent stem cells into vascular endothelial cells using defined culture conditions.The combinatorial use of small molecule compounds, growth factors as well as morphogens directs human pluripotent stem cells toward endothelial cells within 6 days.

Alternative transcription of a shorter, non-anti-angiogenic thrombospondin-2 variant in cancer-associated blood vessels.

Thrombospondin-2 (TSP2) is an anti-angiogenic matricellular protein that inhibits tumor growth and angiogenesis. Tumor-associated blood vascular endothelial cells (BECs) were isolated from human invasive bladder cancers and from matched normal bladder tissue by immuno-laser capture microdissection. Exon expression profiling analyses revealed a particularly high expression of a short TSP2 transcript containing only the last 9 (3') exons of the full-length TSP2 transcript.

Requirements for Using iPSC-Based Cell Models for Assay Development in Drug Discovery.

A prevalent challenge in drug discovery is the translation of findings from preclinical research into clinical success. Currently, more physiological in vitro systems are being developed to overcome some of these challenges. In particular, induced pluripotent stem cells (iPSCs) have provided the opportunity to generate human cell types that can be utilized for developing more disease-relevant cellular assay models.

An integrated expression atlas of miRNAs and their promoters in human and mouse.

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species.

High expression of insulin receptor on tumour-associated blood vessels in invasive bladder cancer predicts poor overall and progression-free survival.

Bladder cancer is a frequently recurring disease with a very poor prognosis once progressed to invasive stages, and tumour-associated blood vessels play a crucial role in this process. In order to identify novel biomarkers associated with progression, we isolated blood vascular endothelial cells (BECs) from human invasive bladder cancers and matched normal bladder tissue, and found that tumour-associated BECs greatly up-regulated the expression of insulin receptor (INSR). High expression of INSR on BECs of invasive bladder cancers was significantly associated with shorter progression-free and overall survival.

Transcriptional profiling of macrophage and tumor cell interactions in vitro.

Macrophages are important mediators of tumor progression and their function is broadly influenced by different microenvironmental stimuli. To understand the molecular basis of the tumor-supporting role of macrophages in aggressive breast cancer we co-cultured human peripheral monocytes with two breast cancer cell lines representing distinct aggressive cellular phenotype and transcriptionally profiled the changes occurring in both cells during in vitro activated crosstalk. Here we provide a detailed description of the experimental design, sample identity and analysis of the Illumina RNA-Seq data, which have been deposited into Gene Expression Omnibus (GEO): GSE75130.

CD73 Predicts Favorable Prognosis in Patients with Nonmuscle-Invasive Urothelial Bladder Cancer.

Aims: CD73 is a membrane associated 5'-ectonucleotidase that has been proposed as prognostic biomarker in various solid tumors. The aim of this study is to evaluate CD73 expression in a cohort of patients with primary bladder cancer in regard to its association with clinicopathological features and disease course.

Methods: Tissue samples from 174 patients with a primary urothelial carcinoma were immunohistochemically assessed on a tissue microarray.

Connexin 43 expression predicts poor progression-free survival in patients with non-muscle invasive urothelial bladder cancer.

Objectives: To evaluate the protein expression of connexin 43 (Cx43) in primary urothelial bladder cancer and test its association with the histopathological characteristics and clinical outcome.

Methods: A tissue microarray containing 348 tissue samples from 174 patients with primary urothelial carcinomas of the bladder was immunohistochemically stained for Cx43. The intensity of staining was semiquantitatively evaluated (score 0, 1+, 2+), and the association with clinicopathological features was assessed.

Characterization of macrophage--cancer cell crosstalk in estrogen receptor positive and triple-negative breast cancer.

Tumor heterogeneity may broadly influence the activation of tumor-associated macrophages. We aimed to dissect how breast cancer cells of different molecular characteristics contribute to macrophage phenotype and function. Therefore, we performed whole transcriptome sequencing of human monocytes that were co-cultured with estrogen receptor positive (ER(+)) or triple-negative (TNBC) breast cancer cell lines and studied the biological responses related to the differential gene activation in both monocytes and cancer cells by pathway analysis.

Steering target selectivity and potency by fragment-based de novo drug design.

Kinase inhibitors: Ligand-based de novo design is validated as a viable technology for rapidly generating innovative compounds possessing the desired biochemical profile. The study discloses the discovery of the most selective vascular endothelial growth factor receptor-2 (VEGFR-2) kinase inhibitor (right in scheme) known to date as prime lead for antiangiogenic drug development.