Liza Botros MD., Manon C. A. Pronk PhD., Jenny Juschten MD., John Liddle, Sofia K. S. H. Morsing, Jaap D. van Buul PhD., Robert H. Bates, Pieter R. Tuinman MD. PhD., Jan S. M. van Bezu, Stephan Huveneers PhD., Harm Jan Bogaard MD. PhD., Victor W. M. van Hinsbergh PhD., Peter L. Hordijk PhD., and Jurjan Aman MD. PhD.

Aims: Endothelial barrier dysfunction leads to edema and vascular leak, carrying high morbidity and mortality. Previously, Abl kinase inhibition was shown to protect against vascular leak. Using the distinct inhibitory profiles of clinically available Abl kinase inhibitors, we aimed to provide a mechanistic basis for novel treatment strategies against vascular leakage syndromes.

Methods & Results: Bosutinib most potently protected against inflammation-induced endothelial barrier disruption. In vivo, bosutinib prevented LPS-induced alveolar protein extravasation in an acute lung injury mice model. Mechanistically, Mitogen-activated Protein 4 Kinase 4 (MAP4K4) was identified as important novel mediator of endothelial permeability, which signals via ezrin, radixin and moesin proteins to increase turnover of integrin-based focal adhesions. The combined inhibition of MAP4K4 and Arg by bosutinib preserved adherens junction integrity and reduced turnover of focal adhesions, which synergistically act to stabilize the endothelial barrier during inflammation.

Conclusion: MAP4K4 was identified as important regulator of endothelial barrier integrity, increasing focal adhesion turnover and disruption of cell-cell junctions during inflammation. Inhibiting both Arg and MAP4K4, the clinically available drug bosutinib may form a viable strategy against vascular leakage syndromes.

Olivia R. Grafinger, Genya Gorshtein, Tyler Stirling, Megan I. Brasher, and Marc G. Coppolino

Malignant cancer cells can invade extracellular matrix (ECM) through the formation of F-actin-rich subcellular structures termed invadopodia. ECM degradation at invadopodia is mediated by matrix metalloproteinases (MMPs), and recent findings indicate that membrane-anchored membrane type 1-matrix metalloproteinase (MT1-MMP) has a primary role in this process. Maintenance of an invasive phenotype is dependent on internalization of MT1-MMP from the plasma membrane and its recycling to sites of ECM remodeling. Internalization of MT1-MMP is dependent on its phosphorylation, and here we examine the role of β1 integrin-mediated signaling in this process. Activation of β1 integrin using the antibody P4G11 induced phosphorylation and internalization of MT1-MMP and resulted in increased cellular invasiveness and invadopodium formation in vitro. We also observed phosphorylation of Src and epidermal growth factor receptor (EGFR) and an increase in their association in response to β1 integrin activation, and determined that Src and EGFR promote phosphorylation of MT1-MMP on Thr⁵⁶⁷. These results suggest that MT1-MMP phosphorylation is regulated by a β1 integrin-Src-EGFR signaling pathway that promotes recycling of MT1-MMP to sites of invadopodia formation during cancer cell invasion.

Osiris Martinez-Guzman, Mathilda M. Willoughby, Arushi Saini, Jonathan V. Dietz, Iryna Bohovych, Amy E. Medlock, Oleh Khalimonchuk, and Amit R. Reddi

Heme is a cofactor and signaling molecule that is essential for much of aerobic life. All heme-dependent processes in eukaryotes require that heme is trafficked from its site of synthesis in the mitochondria to hemoproteins located throughout the cell. However, the mechanisms governing the mobilization of heme out of the mitochondria, and the spatio-temporal dynamics of these processes, are poorly understood. Herein, using genetically encoded fluorescent heme sensors, we developed a live cell assay to monitor heme distribution dynamics between the mitochondrial inner-membrane, where heme is synthesized, and the mitochondrial matrix, cytosol, and nucleus. Surprisingly, heme trafficking to the nucleus is ~25% faster than to the cytosol or mitochondrial matrix, which are nearly identical, potentially supporting a role for heme as a mitochondrial-nuclear retrograde signal. Moreover, we discovered that the heme synthetic enzyme, 5-aminolevulinic acid synthase (ALAS), and GTPases in control of the mitochondrial dynamics machinery, Mgm1 and Dnm1, and ER contact sites, Gem1, regulate the flow of heme between the mitochondria and nucleus. Overall, our results indicate that there are parallel pathways for the distribution of bioavailable heme.

Benjamin Ziman, Peter Karabinis, Paul Barghouth, and Nestor J. Oviedo

Nutrient availability upon feeding leads to an increase in body size in the planarian Schmidtea mediterranea. However, it remains unclear how food consumption integrates with cell division at the organismal level. Here we show that Sirtuins is evolutionarily conserved in planarians and specifically demonstrate that Sirtuin-1 (Smed-Sirt-1) regulates organismal growth by impairing both feeding behavior and intestinal morphology. Disruption of Smed-Sirt-1 with either RNAi or pharmacological treatment leads to reduced animal growth. Conversely, enhancement of Smed-Sirt-1 with resveratrol accelerates growth. Differences in growth rates were associated with changes in the amount of time to locate food and overall consumption. Furthermore, Smed-Sirt-1(RNAi) animals displayed reduced cell death and increased stem cell proliferation accompanied by impaired expression of intestinal lineage progenitors and reduced branching of the gut. Altogether, our findings indicate Sirtuin-1 is a crucial metabolic hub capable of controlling animal behavior, tissue renewal and morphogenesis of the adult intestine.

Xian Hu, Salma Jalal, Michael Sheetz, Oddmund Bakke, and Felix Margadant

Despite progress made in confocal microscopy, even fast systems still have insufficient temporal resolution for detailed live cell volume imaging, such as tracking rapid movement of membrane vesicles in three-dimensional space. Depending on the shortfall, this may result in undersampling and/or motion artifacts that ultimately limit the quality of the imaging data. By sacrificing detailed information in the Z-direction, we propose a new imaging modality that involves capturing fast "projections" from the field of depth which shortens imaging time by approximately an order of magnitude as compared to standard volumetric confocal imaging. With faster imaging, radiation exposure to the sample is reduced, resulting in less fluorophore photobleaching and potential photodamage. The implementation minimally requires two synchronized control signals that drive a piezo stage and trigger the camera exposure. The device generating the signals has been tested on spinning disk confocals and instant structured-illumination-microscopy (iSIM) microscopes. Our calibration images show that the approach provides highly repeatable and stable imaging conditions that enable photometric measurements of the acquired data, in both standard live imaging and super-resolution modes.

Hem Sapkota, Jonathan D. Wren, and Gary J. Gorbsky

Centrosomes focus microtubules to promote mitotic spindle bipolarity, a critical requirement for balanced chromosome segregation. Comprehensive understanding of centrosome function and regulation requires a complete inventory of components. While many centrosome components have been identified, others may yet remain undiscovered. We have used a bioinformatics approach, based on "guilt by association" expression to identify novel mitotic components among the large group of predicted human proteins that have yet to be functionally characterized. Here we identify Chondrosarcoma-Associated Gene 1 (CSAG1) in maintaining centrosome integrity during mitosis. Depletion of CSAG1 disrupts centrosomes and leads to multipolar spindles more effectively in cells with compromised p53 function. Thus, CSAG1 may reflect a class of "mitotic addiction" genes whose expression is more essential in transformed cells.

Björn Eismann, Teresa G. Krieger, Jürgen Beneke, Ruben Bulkescher, Lukas Adam, Holger Erfle, Carl Herrmann, Roland Eils, and Christian Conrad

3D cell cultures enable the in vitro study of dynamic biological processes such as the cell cycle, but their use in high-throughput screens remains impractical with conventional fluorescent microscopy. Here, we present a screening workflow for the automated evaluation of mitotic phenotypes in 3D cell cultures by light-sheet microscopy. After sample preparation by a liquid handling robot, cell spheroids are imaged for 24 hours in toto with a dual-view inverted selective plane illumination microscope (diSPIM) with a much improved signal-to-noise ratio, higher imaging speed, isotropic resolution and reduced light exposure compared to a spinning disc confocal microscope. A dedicated high-content image processing pipeline implements convolutional neural network based phenotype classification. We illustrate the potential of our approach by siRNA knock-down and epigenetic modification of 28 mitotic target genes for assessing their phenotypic role in mitosis. By rendering light-sheet microscopy operational for high-throughput screening applications, this workflow enables target gene characterization or drug candidate evaluation in tissue-like 3D cell culture models.

Su Ming Sun, Amandine Batte, Mireille Tittel-Elmer, Sophie van der Horst, Tibor van Welsem, Gordon Bean, Trey Ideker, Fred van Leeuwen, and Haico van Attikum

Eukaryotic chromosomes are replicated in interphase and the two newly duplicated sister chromatids are held together by the cohesin complex and several cohesin auxiliary factors. Sister chromatid cohesion is essential for accurate chromosome segregation during mitosis, yet has also been implicated in other processes, including DNA damage repair, transcription and DNA replication. To assess how cohesin and associated factors functionally interconnect and coordinate with other cellular processes, we systematically mapped genetic interactions of 17 cohesin genes centered on quantitative growth measurements of >52,000 gene pairs in budding yeast. Integration of synthetic genetic interactions unveiled a cohesin functional map that constitutes 373 genetic interactions, revealing novel functional connections with post-replication repair, microtubule organization and protein folding. Accordingly, we show that the microtubule-associated protein Irc15 and the prefoldin complex members Gim3, Gim4 and Yke2 are new factors involved in sister chromatid cohesion. Our genetic interaction map thus provides a unique resource for further identification and functional interrogation of cohesin proteins. Since mutations in cohesin proteins have been associated with cohesinopathies and cancer, it may also identify cohesin interactions relevant in disease etiology.

Olga Kopach, Noemi Esteras, Selina Wray, Dmitri A. Rusakov, and Andrey Y. Abramov

Frontotemporal dementia and parkinsonism (FTDP-17) caused by the 10+16 splice-site mutation in the MAPT provides an established platform to model tau-related dementia in vitro. Human iPSC-derived neurons have been shown to recapitulate the neurodevelopmental profile of tau pathology during in vitro corticogenesis as in the adult human brain. However, the neurophysiological phenotype of these cells has remained unknown, leaving unanswered questions over the functional relevance and the gnostic power of this disease model. Here we used electrophysiology to explore the membrane properties and intrinsic excitability of the generated neurons to find that human cells mature by ~150 days of neurogenesis to become compatible with matured cortical neurons. In earlier FTDP-17, neurons, however, exhibited a depolarized resting membrane potential associated with increased resistance and reduced voltage-gated Na⁺- and K⁺-channel-mediated conductance. The Na_v1.6 protein was reduced in FTDP-17. These led to a reduced cell capability of induced firing and changed action potential waveform in FTDP-17. The revealed neuropathology may thus contribute to the clinicopathological profile of the disease. This sheds new light on the significance of human models of dementia in vitro.

Julian Chollet, Alexander Dünkler, Anne Bäuerle, Laura Vivero-Pol, Medhanie A. Mulaw, Thomas Gronemeyer, and Nils Johnsson

Yeast cells select the position of their new bud at the beginning of each cell cycle. The recruitment of the septins to this prospective bud site is one of the critical events in a complex assembly pathway that culminates in the outgrowth of a new daughter cell. Hereby, the septin-rods follow the high concentration of Cdc42_GTP that is generated by the focused localization of its GEF Cdc24. We show that shortly before budding Cdc24 not only activates Cdc42 but also transiently interacts with Cdc11, the septin subunit that caps both ends of the septin rods. Mutations in Cdc24 reducing the affinity to Cdc11 impair septin recruitment and decrease the stability of the polarity patch. The interaction between septins and Cdc24 thus reinforces bud assembly at sites where septin structures are formed. Once the septins polymerize into the ring, Cdc24 is found at the cortex of the bud and directs its further outgrowth from this position.

Kelly L. Dunlevy, Valentina Medvedeva, Jade E. Wilson, Mohammed Hoque, Trinity Pellegrin, Adam Maynard, Madison M. Kremp, Jason S. Wasserman, Andrey Poleshko, and Richard A. Katz

A large fraction of epigenetically silent heterochromatin is anchored to the nuclear periphery via "tethering proteins" that function to bridge heterochromatin and the nuclear membrane or nuclear lamina. We identified previously a human tethering protein, PRR14, that binds heterochromatin through an N-terminal domain, but the mechanism and regulation of nuclear lamina association remained to be investigated. Here we identify an evolutionarily conserved PRR14 nuclear lamina binding domain (LBD) that is both necessary and sufficient for positioning of PRR14 at the nuclear lamina. We also show that PRR14 associates dynamically with the nuclear lamina, and provide evidence that such dynamics are regulated through phosphorylation-dephosphorylation of the LBD. Furthermore, we identified a PP2A phosphatase recognition motif within the evolutionarily conserved PRR14 C-terminal Tantalus domain. Disruption of this motif affected PRR14 localization to the nuclear lamina. The overall findings demonstrate a heterochromatin anchoring mechanism whereby the PRR14 tether simultaneously binds heterochromatin and the nuclear lamina through two separable, modular domains. The findings also describe an optimal PRR14 LBD fragment that could be used for efficient targeting of fusion proteins to the nuclear lamina.

Samantha-Su Z. Taylor, Nicole L. Jacobsen, Tasha K. Pontifex, Paul Langlais, and Janis M. Burt

Connexin 37 (Cx37) expression profoundly suppresses proliferation of rat insulinoma (Rin) cells in a manner dependent on gap junction channel (GJCh) functionality and the presence and phosphorylation status of its carboxyl-terminus (CT). In Rin cells growth arrested by induced Cx37 expression, serine 319 (S319) is frequently phosphorylated. Preventing phosphorylation at this site (alanine substitution; S319A) relieved Cx37 of its growth suppressive effect whereas mimicking phosphorylation at this site (aspartate substitution; S319D) enhanced Cx37's growth suppressive properties. Like Cx37-WT, -S319D GJChs and hemichannels (HChs) preferred the closed state, rarely opening fully, and gated slowly. In contrast, Cx37-S319A channels preferred open states, opened fully, and gated rapidly. These data indicate that phosphorylation-dependent conformational differences in Cx37 protein and channel function underlie Cx37-induced growth arrest vs. growth permissive phenotypes. That the closed state of -WT and Cx37-S319D GJChs and HChs favors growth arrest suggests that rather than specific permeants mediating cell cycle arrest, the closed conformation instead supports interaction of Cx37 with growth regulatory proteins that result in growth arrest.

Isabella Guardamagna, Elisabetta Bassi, Monica Savio, Paola Perucca, Ornella Cazzalini, Ennio Prosperi, and Lucia A. Stivala

Assessing DNA repair is an important endpoint to study the DNA damage response for investigating the biochemical mechanisms of this process and the efficacy of chemotherapy, which often uses DNA damaging compounds. Numerous in vitro methods to biochemically characterize DNA repair mechanisms have been developed so far. However, they show some limitations mainly due to the lack of chromatin organization. Here we describe a functional cell-free system to study DNA repair synthesis in vitro, using G1-phase nuclei isolated from human cells treated with different genotoxic agents. Upon incubation in the correspondent damage-activated cytosolic extracts, containing biotin-16-dUTP, nuclei are able to initiate DNA repair synthesis. The use of specific DNA synthesis inhibitors markedly decreased biotinylated dUTP incorporation, indicating the specificity of the repair response. Exogenously added human recombinant PCNA protein, but not the sensors of UV-DNA damage DDB2 or DDB1, stimulated UVC induced dUTP incorporation. In contrast, a DDB2^PCNA- mutant protein, unable to associate with PCNA, interfered with DNA repair synthesis. Given its responsiveness to different type of DNA lesions, this system offers an additional tool to study DNA repair mechanisms.

The geologic faults responsible for the rise of the eastern Andes mountains in Colombia became active 25 million years ago—18 million years before the previously accepted start date for the Andes’ rise.

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The Hope Diamond’s red glow has long been considered a unique property of that stone. Most blue diamonds produce a bluish-white phosphorescence if exposed to ultraviolet light. The few other diamonds known to emit red phosphorescence were commonly assumed to have been from the even larger original stone from which the Hope was cut.

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Smithsonian Institution Libraries has recently acquired a rare first edition of Darwin's Geological Observations on the Volcanic Islands, Visited During the Voyage of the H.M.S. Beagle.

The post With 1844 first edition, Smithsonian Libraries completes its collection of Charles Darwin’s three-volume geology series appeared first on Smithsonian Insider.

Formed some 250,000 years ago when the Nile River pushed through a low channel near Wadi Tushka, it flooded the eastern Sahara, creating a lake that at its highest level covered more than 42,000 square miles.

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To understand the long-term effects of a prolonged tropical storm in the Panama Canal watershed, Robert Stallard, staff scientist at the Smithsonian Tropical Research Institute and research hydrologist at the U.S. Geological Survey, and Armando Ubeda, the LightHawk Mesoamerica program manager, organized four flights over the watershed to create a digital map of landslide scars.

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More than 500 carats of rough diamonds were recently donated to the Department of Mineral Sciences of the Smithsonian’s Natural History Museum by Jewlers Mutual Insurance Co. of Neenah, Wis.

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The new island visible in the satellite photograph is the top of a giant shield volcano located on the rift axis in the Red Sea where the continental plates of Africa and Arabia are pulling apart.

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A mysterious cycle of booms and busts in marine biodiversity over the past 500 million years could be tied to a periodic uplifting of the world's continents, scientists report

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The National Museum of Natural History will permanently display the Dom Pedro Aquamarine, which is the largest single piece of cut-gem aquamarine in the world, beginning Dec. 6.

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Recent airborne geophysical surveys near Decorah, Iowa are providing an unprecedented look at a 470-million-year-old meteorite crater concealed beneath bedrock and sediments. The aerial surveys, […]

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A full 94 percent of the dead zones in the world’s oceans lie in regions expected to warm at least 2 degrees Celsius by the […]

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Forget what you thought you knew about geology. Some minerals can roll up like flaky Belgian piroulines. For the last several decades, mining operations in […]

The post Rolled-Up Mystery Mineral may cause Craving for Piroulines appeared first on Smithsonian Insider.

Everyone knows that the gravitational tugging of the moon makes earth’s oceans swell and recede. But did you know Earth’s tugging is causing the moon […]

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Have an upset stomach? Pop a chalky, chewable antacid. Maybe you’ve got a painful cut or burn. No problem; reach for a healing ointment or […]

The post Glittering, mesmerizing, lifesaving: Hospital exhibit showcases minerals used in medicine appeared first on Smithsonian Insider.

Antarctica is home to fewer insect species than anywhere else on earth. But it wasn’t always that way. A geologist digging in bluffs on Antarctica’s […]

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To the Hopewell Culture, ancient Native Americans who sought out the exotic from near and far, metal was a rare and precious resource. Copper, found […]

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Structural investigations of amorphous and nanocrystalline phases forming in solution are historically challenging. Few methods are capable of in situ atomic structural analysis and rigorous control of the system. A mixed-flow reactor (MFR) is used for total X-ray scattering experiments to examine the short- and long-range structure of phases in situ with pair distribution function (PDF) analysis. The adaptable experimental setup enables data collection for a range of different system chemistries, initial supersaturations and residence times. The age of the sample during analysis is controlled by adjusting the flow rate. Faster rates allow for younger samples to be examined, but if flow is too fast not enough data are acquired to average out excess signal noise. Slower flow rates form older samples, but at very slow speeds particles settle and block flow, clogging the system. Proper background collection and subtraction is critical for data optimization. Overall, this MFR method is an ideal scheme for analyzing the in situ structures of phases that form during crystal growth in solution. As a proof of concept, high-resolution total X-ray scattering data of amorphous and crystalline calcium phosphates and amorphous calcium carbonate were collected for PDF analysis.

Serial crystallography records still diffraction patterns from single, randomly oriented crystals, then merges data from hundreds or thousands of them to form a complete data set. To process the data, the diffraction patterns must first be indexed, equivalent to determining the orientation of each crystal. A novel automatic indexing algorithm is presented, which in tests usually gives significantly higher indexing rates than alternative programs currently available for this task. The algorithm does not require prior knowledge of the lattice parameters but can make use of that information if provided, and also allows indexing of diffraction patterns generated by several crystals in the beam. Cases with a small number of Bragg spots per pattern appear to particularly benefit from the new approach. The algorithm has been implemented and optimized for fast execution, making it suitable for real-time feedback during serial crystallography experiments. It is implemented in an open-source C++ library and distributed under the LGPLv3 licence. An interface to it has been added to the CrystFEL software suite.

In this study, the atomic structure of the ternary icosahedral ZnMgTm quasicrystal (QC) is investigated by means of single-crystal X-ray diffraction. The structure is found to be a member of the Bergman QC family, frequently found in Zn–Mg–rare-earth systems. The ab initio structure solution was obtained by the use of the Superflip software. The infinite structure model was founded on the atomic decoration of two golden rhombohedra, with an edge length of 21.7 Å, constituting the Ammann–Kramer–Neri tiling. The refined structure converged well with the experimental diffraction diagram, with the crystallographic R factor equal to 9.8%. The Bergman clusters were found to be bonded by four possible linkages. Only two linkages, b and c, are detected in approximant crystals and are employed to model the icosahedral QCs in the cluster approach known for the CdYb Tsai-type QC. Additional short b and a linkages are found in this study. Short interatomic distances are not generated by those linkages due to the systematic absence of atoms and the formation of split atomic positions. The presence of four linkages allows the structure to be pictured as a complete covering by rhombic triacontahedral clusters and consequently there is no need to define the interstitial part of the structure (i.e. that outside the cluster). The 6D embedding of the solved structure is discussed for the final verification of the model.

Entangled embedded periodic nets and crystal frameworks are defined, along with their dimension type, homogeneity type, adjacency depth and periodic isotopy type. Periodic isotopy classifications are obtained for various families of embedded nets with small quotient graphs. The 25 periodic isotopy classes of depth-1 embedded nets with a single-vertex quotient graph are enumerated. Additionally, a classification is given of embeddings of n-fold copies of pcu with all connected components in a parallel orientation and n vertices in a repeat unit, as well as demonstrations of their maximal symmetry periodic isotopes. The methodology of linear graph knots on the flat 3-torus [0,1)3 is introduced. These graph knots, with linear edges, are spatial embeddings of the labelled quotient graphs of an embedded net which are associated with its periodicity bases.

A new approach is presented to obtain candidate structures from atomic pair distribution function (PDF) data in a highly automated way. It fetches, from web-based structural databases, all the structures meeting the experimenter's search criteria and performs structure refinements on them without human intervention. It supports both X-ray and neutron PDFs. Tests on various material systems show the effectiveness and robustness of the algorithm in finding the correct atomic crystal structure. It works on crystalline and nanocrystalline materials including complex oxide nanoparticles and nanowires, low-symmetry and locally distorted structures, and complicated doped and magnetic materials. This approach could greatly reduce the traditional structure searching work and enable the possibility of high-throughput real-time auto-analysis PDF experiments in the future.

The generalized Darwin–Hamilton equations [Wuttke (2014). Acta Cryst. A70, 429–440] describe multiple Bragg reflection from a thick, ideally imperfect crystal. These equations are simplified by making full use of energy conservation, and it is demonstrated that the conventional two-ray Darwin–Hamilton equations are obtained as a first-order approximation. Then an efficient numeric solution method is presented, based on a transfer matrix for discretized directional distribution functions and on spectral collocation in the depth coordinate. Example solutions illustrate the orientational spread of multiply reflected rays and the distortion of rocking curves, especially if the detector only covers a finite solid angle.

As you read here
In a 24-hour-period between July 9 and 10, 2009 a clouded leopard cub, a Przewalski's horse, and a red panda cub were all born at Smithsonian's National Zoo's Conservation and Research Center in Front Royal, Virginia.

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Black holes sound wildly complicated. After all, there are all sorts of bizarre things going on: intense gravity, the warping of the fabric of space, the distortion of time itself. But when it comes to describing black holes, it comes down to just two numbers: the mass of the black hole and its spin. That’s right. Everything you physically need to describe a black hole is found in just these two numbers.

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Bruce Campbell, of the Center for Earth and Planetary Studies at the Smithsonian's National Air and Space Museum, is at the National Radio Astronomy Observatory in Green Bank, W. Va., to make a radar map of the Moon.

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Dr. Mary Hagedorn, a marine biologist at the Smithsonian Institution, talks about her research to understand and conserve our oceans' corals.

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Dr. Mary Hagedorn, a marine biologist at the Smithsonian Institution, talks about her research to understand and conserve our oceans' corals. To meet more scientists, visit https://insider.si.edu.

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