STIGMA: Single-cell tissue-specific gene prioritization using machine learning.

Clinical exome and genome sequencing have revolutionized the understanding of human disease genetics. Yet many genes remain functionally uncharacterized, complicating the establishment of causal disease links for genetic variants. While several scoring methods have been devised to prioritize these candidate genes, these methods fall short of capturing the expression heterogeneity across cell subpopulations within tissues.

Single-cell, whole-embryo phenotyping of mammalian developmental disorders.

Mouse models are a critical tool for studying human diseases, particularly developmental disorders. However, conventional approaches for phenotyping may fail to detect subtle defects throughout the developing mouse. Here we set out to establish single-cell RNA sequencing of the whole embryo as a scalable platform for the systematic phenotyping of mouse genetic models.

Single-cell sequencing: promises and challenges for human genetics.

Over the last decade, single-cell sequencing has transformed many fields. It has enabled the unbiased molecular phenotyping of even whole organisms with unprecedented cellular resolution. In the field of human genetics, where the phenotypic consequences of genetic and epigenetic alterations are of central concern, this transformative technology promises to functionally annotate every region in the human genome and all possible variants within them at a massive scale.

Comparative single-cell analysis of the adult heart and coronary vasculature.

The structure and function of the circulatory system, including the heart, have undergone substantial changes with the vertebrate evolution. Although the basic function of the heart is to pump blood through the body, its size, shape, speed, regeneration capacity, etc. vary considerably across species.

Serotonergic regulation of bipolar cell survival in the developing cerebral cortex.

One key factor underlying the functional balance of cortical networks is the ratio of excitatory and inhibitory neurons. The mechanisms controlling the ultimate number of interneurons are beginning to be elucidated, but to what extent similar principles govern the survival of the large diversity of cortical inhibitory cells remains to be investigated. Here, we investigate the mechanisms regulating developmental cell death in neurogliaform cells, bipolar cells, and basket cells, the three main populations of interneurons originating from the caudal ganglionic eminence and the preoptic region.

The role of single-cell genomics in human genetics.

Single-cell sequencing is a powerful approach that can detect genetic alterations and their phenotypic consequences in the context of human development, with cellular resolution. Humans start out as single-cell zygotes and undergo fission and differentiation to develop into multicellular organisms. Before fertilisation and during development, the cellular genome acquires hundreds of mutations that propagate down the cell lineage.

Spindle reorientation in response to mechanical stress is an emergent property of the spindle positioning mechanisms.

Proper orientation of the mitotic spindle plays a crucial role in embryos, during tissue development, and in adults, where it functions to dissipate mechanical stress to maintain tissue integrity and homeostasis. While mitotic spindles have been shown to reorient in response to external mechanical stresses, the subcellular cues that mediate spindle reorientation remain unclear. Here, we used a combination of optogenetics and computational modeling to investigate how mitotic spindles respond to inhomogeneous tension within the actomyosin cortex.

Input-specific control of interneuron numbers in nascent striatal networks.

The assembly of functional neuronal circuits requires appropriate numbers of distinct classes of neurons, but the mechanisms through which their relative proportions are established remain poorly defined. Investigating the mouse striatum, we found that the two most prominent subtypes of striatal interneurons, parvalbumin-expressing (PV+) GABAergic and cholinergic (ChAT+) interneurons, undergo extensive programmed cell death between the first and second postnatal weeks. Remarkably, the survival of PV+ and ChAT+ interneurons is regulated by distinct mechanisms mediated by their specific afferent connectivity.

Transcriptional Alterations in X-Linked Dystonia-Parkinsonism Caused by the SVA Retrotransposon.

X-linked dystonia-parkinsonism (XDP) is a severe neurodegenerative disorder that manifests as adult-onset dystonia combined with parkinsonism. A SINE-VNTR-Alu (SVA) retrotransposon inserted in an intron of the gene reduces its expression and alters splicing in XDP patient-derived cells. As a consequence, increased levels of the intron retention transcript can be found in XDP cells as compared to healthy controls.

Position effects at the FGF8 locus are associated with femoral hypoplasia.

Copy-number variations (CNVs) are a common cause of congenital limb malformations and are interpreted primarily on the basis of their effect on gene dosage. However, recent studies show that CNVs also influence the 3D genome chromatin organization. The functional interpretation of whether a phenotype is the result of gene dosage or a regulatory position effect remains challenging.

Lifetime-Engineered Ruby Nanoparticles (Tau-Rubies) for Multiplexed Imaging of μ-Opioid Receptors.

To address the growing demand for simultaneous imaging of multiple biomarkers in highly scattering media such as organotypic cell cultures, we introduce a new type of photoluminescent nanomaterial termed "tau-ruby" composed of ruby nanocrystals (AlO:Cr) with tunable emission lifetime. The lifetime tuning range from 2.4 to 3.

Influence of FRET and fluorescent protein maturation on the quantification of binding affinity with dual-channel fluorescence cross-correlation spectroscopy.

Protein-protein interactions at the plasma membrane mediate transmembrane signaling. Dual-channel fluorescence cross-correlation spectroscopy (dc-FCCS) is a method with which these interactions can be quantified in a cellular context. However, factors such as incomplete maturation of fluorescent proteins, spectral crosstalk, and fluorescence resonance energy transfer (FRET) affect quantification.

Functionalization and Bioconjugation of Nanoruby for Long-Term, Ultrasensitive Imaging of Μu-Opioid Receptors.

Sensitive and long-term fluorescence imaging of G-protein-coupled receptors enables exploration of molecular level details of these therapeutically relevant proteins, including their expression, localization, signaling, and intracellular trafficking. In this context, labeling these receptors with bright and photostable fluorescent probes is necessary to overcome current imaging problems such as optical background and photobleaching. Here, we describe the procedures to functionalize nanoruby (and other similar nanoparticles) with NeutrAvidin (a streptavidin analog) and to apply this bioconjugate for ultrasensitive, long-term imaging of μ-opioid receptors heterologously expressed in AtT-20 cells.

Intrinsic Efficacy of Opioid Ligands and Its Importance for Apparent Bias, Operational Analysis, and Therapeutic Window.

Evidence from several novel opioid agonists and knockout animals suggests that improved opioid therapeutic window, notably for analgesia versus respiratory depression, is a result of ligand bias downstream of activation of the -opioid receptor (MOR) toward G protein signaling and away from other pathways, such as arrestin recruitment. Here, we argue that published claims of opioid bias based on application of the operational model of agonism are frequently confounded by failure to consider the assumptions of the model. These include failure to account for intrinsic efficacy and ceiling effects in different pathways, distortions introduced by analysis of amplified (G protein) versus linear (arrestin) signaling mechanisms, and nonequilibrium effects in a dynamic signaling cascade.

Pyramidal cell regulation of interneuron survival sculpts cortical networks.

Complex neuronal circuitries such as those found in the mammalian cerebral cortex have evolved as balanced networks of excitatory and inhibitory neurons. Although the establishment of appropriate numbers of these cells is essential for brain function and behaviour, our understanding of this fundamental process is limited. Here we show that the survival of interneurons in mice depends on the activity of pyramidal cells in a critical window of postnatal development, during which excitatory synaptic input to individual interneurons predicts their survival or death.

Electromechanics and Volume Dynamics in Nonexcitable Tissue Cells.

Cell volume regulation is fundamentally important in phenomena such as cell growth, proliferation, tissue homeostasis, and embryogenesis. How the cell size is set, maintained, and changed over a cell's lifetime is not well understood. In this work we focus on how the volume of nonexcitable tissue cells is coupled to the cell membrane electrical potential and the concentrations of membrane-permeable ions in the cell environment.

Challenges and ethical issues in the course of palliative care management for people living with advanced neurologic diseases.

In the recent years, there has been an increase in awareness with regards to the role of palliative care (PC) in management of neurologic diseases. In 1996, the need to incorporate PC in the care for patients with neurologic conditions was recognized by the American Academy of Neurology (AAN) Ethics and Humanities Subcommittee. The gaps in research, education and the ability to deliver adequate PC were then acknowledged by the National Academy of Sciences with their publication of "Approaching death: improving care at the end of life" and most recently, continued goals in improving PC was highlighted by another recent publication "Dying in America: improving quality and honoring individual preferences near the end of life".

Development of Bright and Biocompatible Nanoruby and Its Application to Background-Free Time-Gated Imaging of G-Protein-Coupled Receptors.

At the forefront of developing fluorescent probes for biological imaging applications are enhancements aimed at increasing their brightness, contrast, and photostability, especially toward demanding applications of single-molecule detection. In comparison with existing probes, nanorubies exhibit unlimited photostability and a long emission lifetime (∼4 ms), which enable continuous imaging at single-particle sensitivity in highly scattering and fluorescent biological specimens. However, their wide application as fluorescence probes has so far been hindered by the absence of facile methods for scaled-up high-volume production and molecularly specific targeting.

Human Prestin: A Candidate PE1 Protein Lacking Stringent Mass Spectrometric Evidence?

The evidence that any protein exists in the Human Proteome Project (HPP; protein evidence 1 or PE1) has revolved primarily (although not exclusively) around mass spectrometry (MS) (93% of PE1 proteins have MS evidence in the latest neXtProt release), with robust and stringent, well-curated metrics that have served the community well. This has led to a significant number of proteins still considered "missing" (i.e.

Force Measurements for Cancer Cells.

During cytoskeleton remodeling, cancer cells generate force at the plasma membrane that originates from chemical motors (e.g., actin).

Movement Initiation Signals in Mouse Whisker Motor Cortex.

Frontal cortex plays a central role in the control of voluntary movements, which are typically guided by sensory input. Here, we investigate the function of mouse whisker primary motor cortex (wM1), a frontal region defined by dense innervation from whisker primary somatosensory cortex (wS1). Optogenetic stimulation of wM1 evokes rhythmic whisker protraction (whisking), whereas optogenetic inactivation of wM1 suppresses initiation of whisking.

Parallel pathways from whisker and visual sensory cortices to distinct frontal regions of mouse neocortex.

The spatial organization of mouse frontal cortex is poorly understood. Here, we used voltage-sensitive dye to image electrical activity in the dorsal cortex of awake head-restrained mice. Whisker-deflection evoked the earliest sensory response in a localized region of primary somatosensory cortex and visual stimulation evoked the earliest responses in a localized region of primary visual cortex.