All eyes on a phosphatase in glioma stem cells.

In this issue of JEM, Zhang et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20202669) identify a dependency of glioma stem cells on tyrosine phosphatase activity of EYA2 and a new role for this phosphatase at the centrosome, offering a new therapeutic approach to target mitotic activity.

Label-free detection of DNA hybridization with a compact LSPR-based fiber-optic sensor.

A miniaturized, robust, localized surface plasmon resonance (LSPR)-coupled fiber-optic (FO) nanoprobe providing an integrated and portable solution for detection of DNA hybridization and measurement of DNA concentrations has been demonstrated. The FO nanoprobe was created by constructing arrays of metallic nanostructures on the end facets of optical fibers utilizing nanofabrication technologies, including electron beam lithography and lift-off processes. The LSPR-FO nanoprobe device offers real-time, label-free, and low-sample-volume quantification of single-strand DNA in water with high sensitivity and selectivity, achieving a limit of detection around 10 fM. These results demonstrate the feasibility of the LSPR-FO nanoprobe device as a compact and low-cost biosensor for detection of short-strand DNA.

Identification of proliferative progenitors associated with prominent postnatal growth of the pons.

The pons controls crucial sensorimotor and autonomic functions. In humans, it grows sixfold postnatally and is a site of paediatric gliomas; however, the mechanisms of pontine growth remain poorly understood. We show that the murine pons quadruples in volume postnatally; growth is fastest during postnatal days 0-4 (P0-P4), preceding most myelination. We identify three postnatal proliferative compartments: ventricular, midline and parenchymal. We find no evidence of postnatal neurogenesis in the pons, but each progenitor compartment produces new astroglia and oligodendroglia; the latter expand 10- to 18-fold postnatally, and are derived mostly from the parenchyma. Nearly all parenchymal progenitors at P4 are Sox2(+)Olig2(+), but by P8 a Sox2(-) subpopulation emerges, suggesting a lineage progression from Sox2(+) 'early' to Sox2(-) 'late' oligodendrocyte progenitor. Fate mapping reveals that >90% of adult oligodendrocytes derive from P2-P3 Sox2(+) progenitors. These results demonstrate the importance of postnatal Sox2(+)Olig2(+) progenitors in pontine growth and oligodendrogenesis.

A Dorsal SHH-Dependent Domain in the V-SVZ Produces Large Numbers of Oligodendroglial Lineage Cells in the Postnatal Brain.

Neural stem cells in different locations of the postnatal mouse ventricular-subventricular zone (V-SVZ) generate different subtypes of olfactory bulb (OB) interneurons. High Sonic hedgehog (SHH) signaling in the ventral V-SVZ regulates the production of specific subtypes of neurons destined for the OB. Here we found a transient territory of high SHH signaling in the dorsal V-SVZ beneath the corpus callosum (CC). Using intersectional lineage tracing in neonates to label dorsal radial glial cells (RGCs) expressing the SHH target gene Gli1, we demonstrate that this region produces many CC cells in the oligodendroglial lineage and specific subtypes of neurons in the OB. The number of oligodendroglial cells generated correlated with the levels of SHH signaling. This work identifies a dorsal domain of SHH signaling, which is an important source of oligodendroglial cells for the postnatal mammalian forebrain.

Postnatal growth of the human pons: a morphometric and immunohistochemical analysis.

Despite its critical importance to global brain function, the postnatal development of the human pons remains poorly understood. In the present study, we first performed magnetic resonance imaging (MRI)-based morphometric analyses of the postnatal human pons (0-18 years; n = 6-14/timepoint). Pons volume increased 6-fold from birth to 5 years, followed by continued slower growth throughout childhood. The observed growth was primarily due to expansion of the basis pontis. T2-based MRI analysis suggests that this growth is linked to increased myelination, and histological analysis of myelin basic protein in human postmortem specimens confirmed a dramatic increase in myelination during infancy. Analysis of cellular proliferation revealed many Ki67(+) cells during the first 7 months of life, particularly during the first month, where proliferation was increased in the basis relative to tegmentum. The majority of proliferative cells in the postnatal pons expressed the transcription factor Olig2, suggesting an oligodendrocyte lineage. The proportion of proliferating cells that were Olig2(+) was similar through the first 7 months of life and between basis and tegmentum. The number of Ki67(+) cells declined dramatically from birth to 7 months and further decreased by 3 years, with a small number of Ki67(+) cells observed throughout childhood. In addition, two populations of vimentin/nestin-expressing cells were identified: a dorsal group near the ventricular surface, which persists throughout childhood, and a parenchymal population that diminishes by 7 months and was not evident later in childhood. Together, our data reveal remarkable postnatal growth in the ventral pons, particularly during infancy when cells are most proliferative and myelination increases.

An enhanced LSPR fiber-optic nanoprobe for ultrasensitive detection of protein biomarkers.

A miniaturized, localized surface plasmon resonance (LSPR)-coupled fiber-optic (FO) nanoprobe is reported as a biosensor that is capable of label-free, sensitive detection of a cancer protein biomarker, free prostate specific antigen (f-PSA). The biosensor is based on the LSPR at the reusable dielectric-metallic hybrid interface with a robust, gold nano-disk array at the fiber end facet that is directly fabricated using EBL and metal lift-off process. The f-PSA has been detected with a mouse anti-human PSA monoclonal antibody (mAb) as a specific receptor linked with a self-assembled monolayer at the LSPR-FO facet surfaces. Experimental investigation and data analysis found near field refractive index (RI) sensitivity at ~226 nm/RIU with current LSPR-FO nanoprobe, and demonstrated the lowest limit of detection (LOD) at 100 fg/mL (~3 fM) of f-PSA in PBS solutions. The control experimentation using 5mg/mL bovine serum albumin in PBS and nonspecific surface test shows the excellent specificity and selectivity in the detection of f-PSA in PBS. These results present important progress towards a miniaturized, multifunctional fiber-optic technology that integrates informational communication and sensing function for developing a high performance, label-free, point-of-care (POC) device.

Interference colors of nematic liquid crystal films at different applied voltages and surface anchoring conditions.

This paper presents the calculated and experimental interference colors of liquid crystal (LC) films due to the optical retardation of two orthogonal electromagnetic components at different surface anchoring conditions and applied voltages. We simulate the deformation of LC director using finite element method and convert the calculated colors into sRGB parameters. A gold micro-structure is fabricated and used to control the optical retardation. Polarizing micrographs were collected and compared with the calculated colors.

A reflection-based localized surface plasmon resonance fiber-optic probe for biochemical sensing.

We report the fabrication and characterization of an optical fiber biochemical sensing probe based on localized surface plasmon resonance (LSPR) and spectra reflection. Ordered array of gold nanodots was fabricated on the optical fiber end facet using electron-beam lithography (EBL). We experimentally demonstrated for the first time the blue shift of the LSPR scattering spectrum with respected to the LSPR extinction spectrum, which had been predicted theoretically. High sensitivity [195.72 nm/refractive index unit (RIU)] of this sensor for detecting changes in the bulk refractive indices has been demonstrated. The label-free affinity bio-molecules sensing capability has also been demonstrated using biotin and streptavidin as the receptor and the analyte.

Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1-S6K pathway signaling.

The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls cell growth in response to nutrient availability and growth factors. TORC1 signaling is hyperactive in cancer, and regulators of TORC1 signaling may represent therapeutic targets for human diseases. To identify novel regulators of TORC1 signaling, we performed a genome-scale RNA interference screen on microarrays of Drosophila melanogaster cells expressing human RPS6, a TORC1 effector whose phosphorylated form we detected by immunofluorescence. Our screen revealed that the TORC1-S6K-RPS6 signaling axis is regulated by many subcellular components, including the Class I vesicle coat (COPI), the spliceosome, the proteasome, the nuclear pore, and the translation initiation machinery. Using additional RNAi reagents, we confirmed 70 novel genes as significant on-target regulators of RPS6 phosphorylation, and we characterized them with extensive secondary assays probing various arms of the TORC1 pathways, identifying functional relationships among those genes. We conclude that cell-based microarrays are a useful platform for genome-scale and secondary screening in Drosophila, revealing regulators that may represent drug targets for cancers and other diseases of deregulated TORC1 signaling.

Optical monitoring of anchoring change in vertically aligned thin liquid crystal film for chemical and biological sensor.

A significant advance in sensitivity of liquid-crystal (LC)-based chemical and biological sensors can be achieved by actively monitoring anchoring energy change. We simulate the deformation of a LC director with different anchoring energies using the finite element method and the optical properties of the LC film using the finite-difference time-domain method. Polarizing micrographs are collected and compared with simulated textures. Measurement of optical transmission is used to monitor the anchoring change. Experimental and simulation results both demonstrate the optical method can effectively monitor the surface anchoring change due to the presence of targeted analytes.

E-beam patterned gold nanodot arrays on optical fiber tips for localized surface plasmon resonance biochemical sensing.

Electron beam lithography (EBL) was used to directly pattern periodic gold nanodot arrays on optical fiber tips. Localized surface plasmon resonance of the E-beam patterned gold nanodot arrays on optical fiber tips was utilized for biochemical sensing. The advantage of the optical fiber based localized surface plasmon resonance (LSPR) sensors is the convenience to work with and work in harsh environments. An optical fiber tip LSPR refractive index sensor of 196 nm per refractive index unit (RIU) sensitivity has been demonstrated. The affinity sensing property of the fiber tip sensor was demonstrated using biotin/streptavidin as the receptor/analyte. The detection limit for streptavidin was determined to be 6 pM.

Demonstration of an ultra-wideband optical fiber inline polarizer with metal nano-grid on the fiber tip.

Dramatic increase in the bandwidth of optical fiber inline polarizer can be achieved by using metal nano-grid on the fiber tip. However, high extinction ratio of such fiber polarizer requires high spatial frequency metal nano girds with high aspect ratio on the small area of optical fiber tip. We report the development of a nano-fabrication process on the optical fiber tip, and the design and realization of the first ultra-wideband fiber inline polarization device with Au nano gird fabricated on a single mode optical fiber end face.

Efficient encoding and rapid decoding for interactive visualization of large three-dimensional hyperspectral chemical images.

Interactive visualization of data from a new generation of chemical imaging systems requires coding that is efficient and accessible. New technologies for secondary ion mass spectrometry (SIMS) generate large three-dimensional, hyperspectral datasets with high spatial and spectral resolution. Interactive visualization is important for chemical analysis, but the raw dataset size exceeds the memory capacities of typical current computer systems and is a significant obstacle. This paper reports the development of a lossless coding method that is memory efficient, enabling large SIMS datasets to be held in fast memory, and supports quick access for interactive visualization. The approach provides pixel indexing, as required for chemical imaging applications, and is based on the statistical characteristics of the data. The method uses differential time-of-flight to effect mass-spectral run-length-encoding and uses a scheme for variable-length, byte-unit representations for both mass-spectral time-of-flight and intensity values. Experiments demonstrate high compression rates and fast access.

Utilizing critical angles in sensing partially ordered liquid crystal profile.

This paper investigates a new approach for tracking nematic uniaxial liquid crystal (LC) profile in partially ordered LC based sensors. This approach utilizes measuring critical angles for total internal reflection (TIR) at the interface of optically isotropic and partially ordered LC film. The proposed optical transduction requires measuring of the ordinary critical angle and two extraordinary critical angles in orthogonal directions to report the LC degree of ordering and the director axis orientation.

CellProfiler Analyst: data exploration and analysis software for complex image-based screens.

BACKGROUND: Image-based screens can produce hundreds of measured features for each of hundreds of millions of individual cells in a single experiment. RESULTS: Here, we describe CellProfiler Analyst, open-source software for the interactive exploration and analysis of multidimensional data, particularly data from high-throughput, image-based experiments. CONCLUSION: The system enables interactive data exploration for image-based screens and automated scoring of complex phenotypes that require combinations of multiple measured features per cell.

Flat top liquid crystal tunable filter using coupled Fabry-Perot cavities.

In this paper, a coupled Fabry-Perot cavities filter, using the liquid crystal as the tunable medium, is investigate to achieve tunable flat top filtering performance across the C and L bands. A tandem coupled Fabry-Perot is presented for a tunable passband filter with flat top and minimum ripple in the passband. The overall tuning range of the filter is 172 nm. Several designs are shown with comparable performance to the commercial available 100 GHz fixed single channel filters.

The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1.

The multiprotein mTORC1 protein kinase complex is the central component of a pathway that promotes growth in response to insulin, energy levels, and amino acids and is deregulated in common cancers. We find that the Rag proteins--a family of four related small guanosine triphosphatases (GTPases)--interact with mTORC1 in an amino acid-sensitive manner and are necessary for the activation of the mTORC1 pathway by amino acids. A Rag mutant that is constitutively bound to guanosine triphosphate interacted strongly with mTORC1, and its expression within cells made the mTORC1 pathway resistant to amino acid deprivation. Conversely, expression of a guanosine diphosphate-bound Rag mutant prevented stimulation of mTORC1 by amino acids. The Rag proteins do not directly stimulate the kinase activity of mTORC1, but, like amino acids, promote the intracellular localization of mTOR to a compartment that also contains its activator Rheb.

Regulation of serotonin biosynthesis by the G proteins Galphao and Galphaq controls serotonin signaling in Caenorhabditis elegans.

To analyze mechanisms that modulate serotonin signaling, we investigated how Caenorhabditis elegans regulates the function of serotonergic motor neurons that stimulate egg-laying behavior. Egg laying is inhibited by the G protein Galphao and activated by the G protein Galphaq. We found that Galphao and Galphaq act directly in the serotonergic HSN motor neurons to control egg laying. There, the G proteins had opposing effects on transcription of the tryptophan hydroxylase gene tph-1, which encodes the rate-limiting enzyme for serotonin biosynthesis. Antiserotonin staining confirmed that Galphao and Galphaq antagonistically affect serotonin levels. Altering tph-1 gene dosage showed that small changes in tph-1 expression were sufficient to affect egg-laying behavior. Epistasis experiments showed that signaling through the G proteins has additional tph-1-independent effects. Our results indicate that (1) serotonin signaling is regulated by modulating serotonin biosynthesis and (2) Galphao and Galphaq act in the same neurons to have opposing effects on behavior, in part, by antagonistically regulating transcription of specific genes. Galphao and Galphaq have opposing effects on many behaviors in addition to egg laying and may generally act, as they do in the egg-laying system, to integrate multiple signals and consequently set levels of transcription of genes that affect neurotransmitter release.

PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase.

The heterotrimeric mTORC1 protein kinase nucleates a signaling network that promotes cell growth in response to insulin and becomes constitutively active in cells missing the TSC1 or TSC2 tumor suppressors. Insulin stimulates the phosphorylation of S6K1, an mTORC1 substrate, but it is not known how mTORC1 kinase activity is regulated. We identify PRAS40 as a raptor-interacting protein that binds to mTORC1 in insulin-deprived cells and whose in vitro interaction with mTORC1 is disrupted by high salt concentrations. PRAS40 inhibits cell growth, S6K1 phosphorylation, and rheb-induced activation of the mTORC1 pathway, and in vitro it prevents the great increase in mTORC1 kinase activity induced by rheb1-GTP. Insulin stimulates Akt/PKB-mediated phosphorylation of PRAS40, which prevents its inhibition of mTORC1 in cells and in vitro. We propose that the relative strengths of the rheb- and PRAS40-mediated inputs to mTORC1 set overall pathway activity and that insulin activates mTORC1 through the coordinated regulation of both.

CellProfiler: image analysis software for identifying and quantifying cell phenotypes.

Biologists can now prepare and image thousands of samples per day using automation, enabling chemical screens and functional genomics (for example, using RNA interference). Here we describe the first free, open-source system designed for flexible, high-throughput cell image analysis, CellProfiler. CellProfiler can address a variety of biological questions quantitatively, including standard assays (for example, cell count, size, per-cell protein levels) and complex morphological assays (for example, cell/organelle shape or subcellular patterns of DNA or protein staining).