TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities

Cell-Surface Marker Signatures for the Isolation of Neural Stem Cells, Glia and Neurons Derived from Human Pluripotent Stem Cells

Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184(+)/CD271(-)/CD44(-)/CD24(+) from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184(-)/CD44(-)/CD15(LOW)/CD24(+) and a population of glia that was CD184(+)/CD44(+) were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo.These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations

The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences

Observation of a sudden cessation of a very-high-energy gamma-ray flare in PKS 1510-089 with H.E.S.S. and MAGIC in May 2016

The flat spectrum radio quasar (FSRQ) PKS 1510-089 is known for its complex multiwavelength behavior, and is one of only a few FSRQs detected at very high energy (VHE, E >100 GeV) -rays. VHE -ray observations with H.E.S.S. and MAGIC during late May and early June 2016 resulted in the detection of an unprecedented flare, which reveals for the first time VHE -ray intranight variability in this source. While a common variability timescale of 1.5 hr is found, there is a significant deviation near the end of the flare with a timescale of ∼ 20 min marking the cessation of the event. The peak flux is nearly two orders of magnitude above the low-level emission. For the first time, curvature is detected in the VHE -ray spectrum of PKS 1510-089, which is fully explained through absorption by the extragalactic background light. Optical R-band observations with ATOM reveal a counterpart of the -ray flare, even though the detailed flux evolution differs from the VHE lightcurve. Interestingly, a steep flux decrease is observed at the same time as the cessation of the VHE flare. In the high energy (HE, E >100 MeV) -ray band only a moderate flux increase is observed with Fermi-LAT, while the HE -ray spectrum significantly hardens up to a photon index of 1.6. A search for broad-line region (BLR) absorption features in the -ray spectrum indicates that the emission region is located outside of the BLR. Radio VLBI observations reveal a fast moving knot interacting with a standing jet feature around the time of the flare. As the standing feature is located ∼ 50 pc from the black hole, the emission region of the flare may have been located at a significant distance from the black hole. If this correlation is indeed true, VHE rays have been produced far down the jet where turbulent plasma crosses a standing shock.Accepted manuscrip

The Speech-Associated Attitude of Children Who Do and Do Not Stutter and the Differential Effect of Age

Fifty-five Flemish children, ages 6 to 13, who stuttered and 55 who did not were the subjects of a two (group) by eight (age) factorial investigation of their response to a Dutch translation of the Communication Attitude Test (C.A.T.). The main effect results confirmed previous C.A.T. findings that, as early as age 6, children who stutter exhibit significantly more in the way of a negative speech-associated attitude than their peers do. In addition, the between-group difference in attitude diverged with age. The C.A.T. scores increased for those who stuttered and decreased for the normally fluent children. These data suggest that the attitude of the two groups of children was differentially affected by their speech-related experience history. It follows from this, and the other findings of the study, that the attitude toward speech of children who stutter warrants early clinical consideration and attention