1
Proc Natl Acad Sci U S A
. 2020 Jun 8;202001270. doi: 10.1073/pnas.2001270117. Online ahead of print.
GPCR-dependent Biasing of GIRK Channel Signaling Dynamics by RGS6 in Mouse Sinoatrial Nodal Cells
Allison Anderson 1, Ikuo Masuho 2, Ezequiel Marron Fernandez de Velasco 1, Atsushi Nakano 3, Lutz Birnbaumer 4 5, Kirill A Martemyanov 2, Kevin Wickman 6
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PMID: 32513692 DOI: 10.1073/pnas.2001270117
Abstract
How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M2R) and adenosine (A1R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M2R and A1R activate a shared pool of cardiac G protein-gated inwardly rectifying K+ (GIRK) channels in SAN cells from adult mice, but A1R-GIRK responses are smaller and slower than M2R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in SAN cells, suppressing M2R-GIRK coupling efficiency and kinetics and A1R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gαo over Gαi as a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαo or Gαi2 was consistent with these findings. Gαi2 ablation had minimal impact on M2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαo ablation decreased the amplitude and slowed the kinetics of M2R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A1R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gαo substrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.

Keywords: G protein; Kir3; adenosine; heart rate; muscarinic.

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The authors declare no competing interest.

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2
Proc Natl Acad Sci U S A
. 2020 Jun 8;202005475. doi: 10.1073/pnas.2005475117. Online ahead of print.
Inequality in Socially Permissible Consumption
Serena F Hagerty 1, Kate Barasz 2
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PMID: 32513745 DOI: 10.1073/pnas.2005475117
Abstract
Lower-income individuals are frequently criticized for their consumption decisions; this research examines why. Eleven preregistered studies document systematic differences in permissible consumption-interpersonal judgments about what is acceptable (or not) for others to consume-such that lower-income individuals' decisions are subject to more negative and restrictive evaluations. Indeed, the same consumption decisions may be deemed less permissible for a lower-income individual than for an individual with higher or unknown income (studies 1A and 1B), even when purchased with windfall funds. This gap persists among participants from a large, nationally representative sample (study 2) and when testing a broad array of "everyday" consumption items (study 3). Additional studies investigate why: The same items are often perceived as less necessary for lower- (versus higher-) income individuals (studies 4 and 5). Combining both permissibility and perceived necessity, additional studies (studies 6 and 7) demonstrate a causal link between the two constructs: A purchase decision will be deemed permissible (or not) to the extent that it is perceived as necessary (or not). However, because-for lower-income individuals-fewer items are perceived as necessary, fewer are therefore socially permissible to consume. This finding not only exposes a fraught double standard, but also portends consequential behavioral implications: People prefer to allocate strictly "necessary" items to lower-income recipients (study 8), even if such items are objectively and subjectively less valuable (studies 9A and 9B), which may result in an imbalanced and inefficient provision of resources to the poor.

Keywords: consumption; economic inequality; income; interpersonal judgments.

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The authors declare no competing interest.

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3
Proc Natl Acad Sci U S A
. 2020 Jun 8;202001064. doi: 10.1073/pnas.2001064117. Online ahead of print.
Three Dimensions of Scientific Impact
Grzegorz Siudem 1, Barbara Żogała-Siudem 2, Anna Cena 3, Marek Gagolewski 2 3 4
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PMID: 32513724 DOI: 10.1073/pnas.2001064117
Abstract
The growing popularity of bibliometric indexes (whose most famous example is the h index by J. E. Hirsch [J. E. Hirsch, Proc. Natl. Acad. Sci. U.S.A. 102, 16569-16572 (2005)]) is opposed by those claiming that one's scientific impact cannot be reduced to a single number. Some even believe that our complex reality fails to submit to any quantitative description. We argue that neither of the two controversial extremes is true. By assuming that some citations are distributed according to the rich get richer rule (success breeds success, preferential attachment) while some others are assigned totally at random (all in all, a paper needs a bibliography), we have crafted a model that accurately summarizes citation records with merely three easily interpretable parameters: productivity, total impact, and how lucky an author has been so far.

Keywords: bibliometric indexes; rich get richer; science of science; scientometrics.

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The authors declare no competing interest.

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4
Proc Natl Acad Sci U S A
. 2020 Jun 8;201922626. doi: 10.1073/pnas.1922626117. Online ahead of print.
Molecular Height Measurement by Cell Surface Optical Profilometry (CSOP)
Sungmin Son 1, Sho C Takatori 1, Brian Belardi 1, Marija Podolski 1, Matthew H Bakalar 1, Daniel A Fletcher 2 3 4
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PMID: 32513731 DOI: 10.1073/pnas.1922626117
Abstract
The physical dimensions of proteins and glycans on cell surfaces can critically affect cell function, for example, by preventing close contact between cells and limiting receptor accessibility. However, high-resolution measurements of molecular heights on native cell membranes have been difficult to obtain. Here we present a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of cell surface molecules and determining their separation by radially averaging across many molecules. We use this method, which we call cell surface optical profilometry (CSOP), to quantify the height of key multidomain proteins on a model cell, as well as to capture average protein and glycan heights on native cell membranes. We show that average height of a protein is significantly smaller than its contour length, due to thermally driven bending and rotation on the membrane, and that height strongly depends on local surface and solution conditions. We find that average height increases with cell surface molecular crowding but decreases with solution crowding by solutes, both of which we confirm with molecular dynamics simulations. We also use experiments and simulations to determine the height of an epitope, based on the location of an antibody, which allows CSOP to profile various proteins and glycans on a native cell surface using antibodies and lectins. This versatile method for profiling cell surfaces has the potential to advance understanding of the molecular landscape of cells and the role of the molecular landscape in cell function.

Keywords: CD45; antibodies; cell surface topography; plasma membrane; superresolution microscopy.

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The authors declare no competing interest.

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5
Proc Natl Acad Sci U S A
. 2020 Jun 8;201922563. doi: 10.1073/pnas.1922563117. Online ahead of print.
Nanoscale Co-Organization and Coactivation of AMPAR, NMDAR, and mGluR at Excitatory Synapses
Julia Goncalves 1 2, Tomas M Bartol 3, Côme Camus 1 2, Florian Levet 1 2 4, Ana Paula Menegolla 1 2, Terrence J Sejnowski 3, Jean-Baptiste Sibarita 1 2, Michel Vivaudou 5 6, Daniel Choquet 1 2 4, Eric Hosy 7 2
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PMID: 32513712 DOI: 10.1073/pnas.1922563117
Abstract
The nanoscale co-organization of neurotransmitter receptors facing presynaptic release sites is a fundamental determinant of their coactivation and of synaptic physiology. At excitatory synapses, how endogenous AMPARs, NMDARs, and mGluRs are co-organized inside the synapse and their respective activation during glutamate release are still unclear. Combining single-molecule superresolution microscopy, electrophysiology, and modeling, we determined the average quantity of each glutamate receptor type, their nanoscale organization, and their respective activation. We observed that NMDARs form a unique cluster mainly at the center of the PSD, while AMPARs segregate in clusters surrounding the NMDARs. mGluR5 presents a different organization and is homogenously dispersed at the synaptic surface. From these results, we build a model predicting the synaptic transmission properties of a unitary synapse, allowing better understanding of synaptic physiology.

Keywords: glutamate receptors; superresolution microscopy; synaptic transmission.

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The authors declare no competing interest.

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6
Proc Natl Acad Sci U S A
. 2020 Jun 8;202003907. doi: 10.1073/pnas.2003907117. Online ahead of print.
Identification of Chondritic Krypton and Xenon in Yellowstone Gases and the Timing of Terrestrial Volatile Accretion
Michael W Broadley 1, Peter H Barry 2, David V Bekaert 3, David J Byrne 3, Antonio Caracausi 4, Christopher J Ballentine 5, Bernard Marty 3
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PMID: 32513744 DOI: 10.1073/pnas.2003907117
Free article
Abstract
Identifying the origin of noble gases in Earth's mantle can provide crucial constraints on the source and timing of volatile (C, N, H2O, noble gases, etc.) delivery to Earth. It remains unclear whether the early Earth was able to directly capture and retain volatiles throughout accretion or whether it accreted anhydrously and subsequently acquired volatiles through later additions of chondritic material. Here, we report high-precision noble gas isotopic data from volcanic gases emanating from, in and around, the Yellowstone caldera (Wyoming, United States). We show that the He and Ne isotopic and elemental signatures of the Yellowstone gas requires an input from an undegassed mantle plume. Coupled with the distinct ratio of 129Xe to primordial Xe isotopes in Yellowstone compared with mid-ocean ridge basalt (MORB) samples, this confirms that the deep plume and shallow MORB mantles have remained distinct from one another for the majority of Earth's history. Krypton and xenon isotopes in the Yellowstone mantle plume are found to be chondritic in origin, similar to the MORB source mantle. This is in contrast with the origin of neon in the mantle, which exhibits an isotopic dichotomy between solar plume and chondritic MORB mantle sources. The co-occurrence of solar and chondritic noble gases in the deep mantle is thought to reflect the heterogeneous nature of Earth's volatile accretion during the lifetime of the protosolar nebula. It notably implies that the Earth was able to retain its chondritic volatiles since its earliest stages of accretion, and not only through late additions.

Keywords: Yellowstone; accretion; mantle plume; noble gases; origin of Earth’s volatiles.

Copyright © 2020 the Author(s). Published by PNAS.

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The authors declare no competing interest.

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7
Proc Natl Acad Sci U S A
. 2020 Jun 8;201921618. doi: 10.1073/pnas.1921618117. Online ahead of print.
PEBP1 Acts as a Rheostat Between Prosurvival Autophagy and Ferroptotic Death in Asthmatic Epithelial Cells
Jinming Zhao 1 2, Haider H Dar 1, Yanhan Deng 1, Claudette M St Croix 3, Zhipeng Li 1, Yoshinori Minami 1, Indira H Shrivastava 1 4, Yulia Y Tyurina 1, Emily Etling 1, Joel C Rosenbaum 5, Tadao Nagasaki 1, John B Trudeau 1, Simon C Watkins 3, Ivet Bahar 4, Hülya Bayır 1 6, Andy P VanDemark 5, Valerian E Kagan 1 7 8 9, Sally E Wenzel 10 2 11 12
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PMID: 32513718 DOI: 10.1073/pnas.1921618117
Abstract
Temporally harmonized elimination of damaged or unnecessary organelles and cells is a prerequisite of health. Under Type 2 inflammatory conditions, human airway epithelial cells (HAECs) generate proferroptotic hydroperoxy-arachidonoyl-phosphatidylethanolamines (HpETE-PEs) as proximate death signals. Production of 15-HpETE-PE depends on activation of 15-lipoxygenase-1 (15LO1) in complex with PE-binding protein-1 (PEBP1). We hypothesized that cellular membrane damage induced by these proferroptotic phospholipids triggers compensatory prosurvival pathways, and in particular autophagic pathways, to prevent cell elimination through programmed death. We discovered that PEBP1 is pivotal to driving dynamic interactions with both proferroptotic 15LO1 and the autophagic protein microtubule-associated light chain-3 (LC3). Further, the 15LO1-PEBP1-generated ferroptotic phospholipid, 15-HpETE-PE, promoted LC3-I lipidation to stimulate autophagy. This concurrent activation of autophagy protects cells from ferroptotic death and release of mitochondrial DNA. Similar findings are observed in Type 2 Hi asthma, where high levels of both 15LO1-PEBP1 and LC3-II are seen in HAECs, in association with low bronchoalveolar lavage fluid mitochondrial DNA and more severe disease. The concomitant activation of ferroptosis and autophagy by 15LO1-PEBP1 complexes and their hydroperoxy-phospholipids reveals a pathobiologic pathway relevant to asthma and amenable to therapeutic targeting.

Keywords: asthma; autophagy; ferroptosis.

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The authors declare no competing interest.

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8
Proc Natl Acad Sci U S A
. 2020 Jun 9;117(23):12550-12557. doi: 10.1073/pnas.1821672117.
Energy Storage Emerging: A Perspective From the Joint Center for Energy Storage Research
Lynn Trahey 1, Fikile R Brushett 2 3, Nitash P Balsara 1 4 5, Gerbrand Ceder 1 5 6, Lei Cheng 1 7, Yet-Ming Chiang 1 8, Nathan T Hahn 1 9, Brian J Ingram 1 10, Shelley D Minteer 1 11, Jeffrey S Moore 1 12 13 14, Karl T Mueller 1 15, Linda F Nazar 1 16 17, Kristin A Persson 1 6 18, Donald J Siegel 1 19, Kang Xu 1 20, Kevin R Zavadil 1 9, Venkat Srinivasan 1 21, George W Crabtree 1 22
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PMID: 32513683 DOI: 10.1073/pnas.1821672117
Abstract
Energy storage is an integral part of modern society. A contemporary example is the lithium (Li)-ion battery, which enabled the launch of the personal electronics revolution in 1991 and the first commercial electric vehicles in 2010. Most recently, Li-ion batteries have expanded into the electricity grid to firm variable renewable generation, increasing the efficiency and effectiveness of transmission and distribution. Important applications continue to emerge including decarbonization of heavy-duty vehicles, rail, maritime shipping, and aviation and the growth of renewable electricity and storage on the grid. This perspective compares energy storage needs and priorities in 2010 with those now and those emerging over the next few decades. The diversity of demands for energy storage requires a diversity of purpose-built batteries designed to meet disparate applications. Advances in the frontier of battery research to achieve transformative performance spanning energy and power density, capacity, charge/discharge times, cost, lifetime, and safety are highlighted, along with strategic research refinements made by the Joint Center for Energy Storage Research (JCESR) and the broader community to accommodate the changing storage needs and priorities. Innovative experimental tools with higher spatial and temporal resolution, in situ and operando characterization, first-principles simulation, high throughput computation, machine learning, and artificial intelligence work collectively to reveal the origins of the electrochemical phenomena that enable new means of energy storage. This knowledge allows a constructionist approach to materials, chemistries, and architectures, where each atom or molecule plays a prescribed role in realizing batteries with unique performance profiles suitable for emergent demands.

Keywords: Joint Center for Energy Storage Research; batteries; energy storage; grid; transportation.

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The authors declare no competing interest.

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9
Proc Natl Acad Sci U S A
. 2020 Jun 8;201917371. doi: 10.1073/pnas.1917371117. Online ahead of print.
ALS/FTD Mutations in UBQLN2 Impede Autophagy by Reducing Autophagosome Acidification Through Loss of Function
Josephine J Wu 1, Ashley Cai 1, Jessie E Greenslade 1, Nicole R Higgins 1, Cong Fan 1, Nhat T T Le 1, Micaela Tatman 1, Alexandra M Whiteley 2, Miguel A Prado 2, Birger V Dieriks 3 4, Maurice A Curtis 3 4, Christopher E Shaw 5 6 7, Teepu Siddique 8, Richard L M Faull 3 4, Emma L Scotter 4 9, Daniel Finley 2, Mervyn J Monteiro 10
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PMID: 32513711 DOI: 10.1073/pnas.1917371117
Abstract
Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.

Keywords: UBQLN2; amyotrophic lateral sclerosis; autophagy; ubiquilin; vacuolar ATPase pump.

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The authors declare no competing interest.

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10
Proc Natl Acad Sci U S A
. 2020 Jun 8;201917259. doi: 10.1073/pnas.1917259117. Online ahead of print.
Multi-omics Analysis on an Agroecosystem Reveals the Significant Role of Organic Nitrogen to Increase Agricultural Crop Yield
Yasunori Ichihashi 1 2, Yasuhiro Date 3 4, Amiu Shino 3, Tomoko Shimizu 3, Arisa Shibata 3, Kie Kumaishi 5, Fumiaki Funahashi 6, Kenji Wakayama 6, Kohei Yamazaki 7, Akio Umezawa 8, Takumi Sato 5, Makoto Kobayashi 3, Mayu Kamimura 9, Miyako Kusano 3 10, Fang-Sik Che 9, Martin O Brien 11, Keitaro Tanoi 11, Makoto Hayashi 3, Ryuhei Nakamura 8, Ken Shirasu 3 12, Jun Kikuchi 3 4 13, Naoto Nihei 14
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PMID: 32513689 DOI: 10.1073/pnas.1917259117
Free article
Abstract
Both inorganic fertilizer inputs and crop yields have increased globally, with the concurrent increase in the pollution of water bodies due to nitrogen leaching from soils. Designing agroecosystems that are environmentally friendly is urgently required. Since agroecosystems are highly complex and consist of entangled webs of interactions between plants, microbes, and soils, identifying critical components in crop production remain elusive. To understand the network structure in agroecosystems engineered by several farming methods, including environmentally friendly soil solarization, we utilized a multiomics approach on a field planted with Brassica rapa We found that the soil solarization increased plant shoot biomass irrespective of the type of fertilizer applied. Our multiomics and integrated informatics revealed complex interactions in the agroecosystem showing multiple network modules represented by plant traits heterogeneously associated with soil metabolites, minerals, and microbes. Unexpectedly, we identified soil organic nitrogen induced by soil solarization as one of the key components to increase crop yield. A germ-free plant in vitro assay and a pot experiment using arable soils confirmed that specific organic nitrogen, namely alanine and choline, directly increased plant biomass by acting as a nitrogen source and a biologically active compound. Thus, our study provides evidence at the agroecosystem level that organic nitrogen plays a key role in plant growth.

Keywords: Brassica rapa; agroecosystem; multiomics; organic nitrogen; soil solarization.

Copyright © 2020 the Author(s). Published by PNAS.

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The authors declare no competing interest.

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11
Proc Natl Acad Sci U S A
. 2020 Jun 8;202005638. doi: 10.1073/pnas.2005638117. Online ahead of print.
Revealing the Three-Dimensional Structure of Liquids Using Four-Point Correlation Functions
Zhen Zhang 1, Walter Kob 2 3
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PMID: 32513730 DOI: 10.1073/pnas.2005638117
Abstract
Disordered systems like liquids, gels, glasses, or granular materials are not only ubiquitous in daily life and in industrial applications, but they are also crucial for the mechanical stability of cells or the transport of chemical and biological agents in living organisms. Despite the importance of these systems, their microscopic structure is understood only on a rudimentary level, thus in stark contrast to the case of gases and crystals. Since scattering experiments and analytical calculations usually give only structural information that is spherically averaged, the three-dimensional (3D) structure of disordered systems is basically unknown. Here, we introduce a simple method that allows probing of the 3D structure of such systems. Using computer simulations, we find that hard sphere-like liquids have on intermediate and large scales a simple structural order given by alternating layers with icosahedral and dodecahedral symmetries, while open network liquids like silica have a structural order with tetrahedral symmetry. These results show that liquids have a highly nontrivial 3D structure and that this structural information is encoded in nonstandard correlation functions.

Keywords: Lennard–Jones; computer simulations; liquids; silica; three-dimensional structure.

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The authors declare no competing interest.

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12
Proc Natl Acad Sci U S A
. 2020 Jun 8;202001849. doi: 10.1073/pnas.2001849117. Online ahead of print.
Cryogenic Single-Molecule Fluorescence Annotations for Electron Tomography Reveal in Situ Organization of Key Proteins in Caulobacter
Peter D Dahlberg 1, Saumya Saurabh 2, Annina M Sartor 1, Jiarui Wang 1 2, Patrick G Mitchell 3, Wah Chiu 3 4, Lucy Shapiro 2, W E Moerner 5
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PMID: 32513734 DOI: 10.1073/pnas.2001849117
Free article
Abstract
Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of ∼30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.

Keywords: CIASM; CLEM; correlative microscopy; cryogenic electron tomography; superresolution.

Copyright © 2020 the Author(s). Published by PNAS.

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The authors declare no competing interest.

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13
Proc Natl Acad Sci U S A
. 2020 Jun 8;201922190. doi: 10.1073/pnas.1922190117. Online ahead of print.
Origin of Interannual Variability in Global Mean Sea Level
Benjamin D Hamlington 1, Christopher G Piecuch 2, John T Reager 3, Hrishi Chandanpurkar 3, Thomas Frederikse 3, R Steven Nerem 4, John T Fasullo 5, Se-Hyeon Cheon 6
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PMID: 32513709 DOI: 10.1073/pnas.1922190117
Abstract
The two dominant drivers of the global mean sea level (GMSL) variability at interannual timescales are steric changes due to changes in ocean heat content and barystatic changes due to the exchange of water mass between land and ocean. With Gravity Recovery and Climate Experiment (GRACE) satellites and Argo profiling floats, it has been possible to measure the relative steric and barystatic contributions to GMSL since 2004. While efforts to "close the GMSL budget" with satellite altimetry and other observing systems have been largely successful with regards to trends, the short time period covered by these records prohibits a full understanding of the drivers of interannual to decadal variability in GMSL. One particular area of focus is the link between variations in the El Niño-Southern Oscillation (ENSO) and GMSL. Recent literature disagrees on the relative importance of steric and barystatic contributions to interannual to decadal variability in GMSL. Here, we use a multivariate data analysis technique to estimate variability in barystatic and steric contributions to GMSL back to 1982. These independent estimates explain most of the observed interannual variability in satellite altimeter-measured GMSL. Both processes, which are highly correlated with ENSO variations, contribute about equally to observed interannual GMSL variability. A theoretical scaling analysis corroborates the observational results. The improved understanding of the origins of interannual variability in GMSL has important implications for our understanding of long-term trends in sea level, the hydrological cycle, and the planet's radiation imbalance.

Keywords: climate variability; global mean sea level; satellite altimetry; sea level.

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The authors declare no competing interest.

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14
Proc Natl Acad Sci U S A
. 2020 Jun 8;202005924. doi: 10.1073/pnas.2005924117. Online ahead of print.
A Long Noncoding RNA Regulates Inflammation Resolution by Mouse Macrophages Through Fatty Acid Oxidation Activation
Yukiteru Nakayama 1, Katsuhito Fujiu 1, Ryuzaburo Yuki 2 3, Yumiko Oishi 3, Masaki Suimye Morioka 1, Takayuki Isagawa 4, Jun Matsuda 1, Tsukasa Oshima 1, Takumi Matsubara 1, Junichi Sugita 1, Fujimi Kudo 2, Atsushi Kaneda 5, Yusuke Endo 6 7, Toshinori Nakayama 6, Ryozo Nagai 8, Issei Komuro 1, Ichiro Manabe 9
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PMID: 32513690 DOI: 10.1073/pnas.2005924117
Abstract
Proper resolution of inflammation is vital for repair and restoration of homeostasis after tissue damage, and its dysregulation underlies various noncommunicable diseases, such as cardiovascular and metabolic diseases. Macrophages play diverse roles throughout initial inflammation, its resolution, and tissue repair. Differential metabolic reprogramming is reportedly required for induction and support of the various macrophage activation states. Here we show that a long noncoding RNA (lncRNA), lncFAO, contributes to inflammation resolution and tissue repair in mice by promoting fatty acid oxidation (FAO) in macrophages. lncFAO is induced late after lipopolysaccharide (LPS) stimulation of cultured macrophages and in Ly6Chi monocyte-derived macrophages in damaged tissue during the resolution and reparative phases. We found that lncFAO directly interacts with the HADHB subunit of mitochondrial trifunctional protein and activates FAO. lncFAO deletion impairs resolution of inflammation related to endotoxic shock and delays resolution of inflammation and tissue repair in a skin wound. These results demonstrate that by tuning mitochondrial metabolism, lncFAO acts as a node of immunometabolic control in macrophages during the resolution and repair phases of inflammation.

Keywords: Long noncoding RNA; inflammation; macrophage.

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The authors declare no competing interest.

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15
Proc Natl Acad Sci U S A
. 2020 Jun 8;201908183. doi: 10.1073/pnas.1908183117. Online ahead of print.
Coupling of Ca 2+ and Voltage Activation in BK Channels Through the αB Helix/Voltage Sensor Interface
Yanyan Geng 1, Zengqin Deng 2, Guohui Zhang 3, Gonzalo Budelli 4, Alice Butler 4, Peng Yuan 2, Jianmin Cui 3, Lawrence Salkoff 4 5, Karl L Magleby 6
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PMID: 32513714 DOI: 10.1073/pnas.1908183117
Abstract
Large-conductance Ca2+ and voltage-activated K+ (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is composed of a voltage sensor domain (VSD), a central pore-gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+ sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+ activations interact, less is known about the mechanisms involved. We explore here these mechanisms by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the Horrigan, Cui, and Aldrich model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+ activation for both Ca2+ bowl and RCK1 Ca2+ sites, suggesting that both high-affinity Ca2+ sites transduce their effect, at least in part, through the αB helix. Mg2+ activation also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to wild type, without other notable differences in the CTD, indicating that structural changes from the mutation were confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca2+ binding and voltage depolarization to pore opening and that shared Ca2+ and voltage transduction pathways involving the αB helix may be involved.

Keywords: BK channel; Ca2+-activated K+ channel; Slo1 channel; allosteric coupling; patch clamp.

Conflict of interest statement
The authors declare no competing interest.

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16
Proc Natl Acad Sci U S A
. 2020 Jun 8;201922294. doi: 10.1073/pnas.1922294117. Online ahead of print.
The Limits of Color Awareness During Active, Real-World Vision
Michael A Cohen 1 2, Thomas L Botch 3, Caroline E Robertson 4
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PMID: 32513698 DOI: 10.1073/pnas.1922294117
Free article
Abstract
Color ignites visual experience, imbuing the world with meaning, emotion, and richness. As soon as an observer opens their eyes, they have the immediate impression of a rich, colorful experience that encompasses their entire visual world. Here, we show that this impression is surprisingly inaccurate. We used head-mounted virtual reality (VR) to place observers in immersive, dynamic real-world environments, which they naturally explored via saccades and head turns. Meanwhile, we monitored their gaze with in-headset eye tracking and then systematically altered the visual environments such that only the parts of the scene they were looking at were presented in color and the rest of the scene (i.e., the visual periphery) was entirely desaturated. We found that observers were often completely unaware of these drastic alterations to their visual world. In the most extreme case, almost a third of observers failed to notice when less than 5% of the visual display was presented in color. This limitation on perceptual awareness could not be explained by retinal neuroanatomy or previous studies of peripheral visual processing using more traditional psychophysical approaches. In a second study, we measured color detection thresholds using a staircase procedure while a set of observers intentionally attended to the periphery. Still, we found that observers were unaware when a large portion of their field of view was desaturated. Together, these results show that during active, naturalistic viewing conditions, our intuitive sense of a rich, colorful visual world is largely incorrect.

Keywords: attention; color; scenes; virtual reality; vision.

Copyright © 2020 the Author(s). Published by PNAS.

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The authors declare no competing interest.

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17
Proc Natl Acad Sci U S A
. 2020 Jun 8;202002861. doi: 10.1073/pnas.2002861117. Online ahead of print.
Gender Differences in the Pathways to Higher Education
Gijsbert Stoet 1, David C Geary 2
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PMID: 32513710 DOI: 10.1073/pnas.2002861117
Free article
Abstract
It is well known that far fewer men than women enroll in tertiary education in the United States and other Western nations. Developed nations vary in the degree to which men are underrepresented, but the Organization for Economic Co-operation and Development (OECD) average lies around 45% male students. We use data from the OECD Education at a Glance statistical reports, the Program for International Student Assessment (PISA), and the World Values Survey to explain the degree to which men are underrepresented. Using a multiple regression model, we show that the combination of both the national reading proficiency levels of 15-y-old boys and girls and the social attitudes toward girls attending university can predict the enrollment in tertiary education 5 y later. The model also shows that parity in some countries is a result of boys' poor reading proficiency and negative social attitudes toward girls' education, which suppresses college enrollment in both sexes, but for different reasons. True equity will at the very least require improvement in boys' reading competencies and the liberalization of attitudes regarding women's pursuit of higher education. At this time, there is little reason to expect that the enrollment gap will decrease, given the stagnating reading competencies in most countries.

Keywords: discrimination; education; gender gap; reading.

Copyright © 2020 the Author(s). Published by PNAS.

Conflict of interest statement
The authors declare no competing interest.

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18
Proc Natl Acad Sci U S A
. 2020 Jun 8;202000950. doi: 10.1073/pnas.2000950117. Online ahead of print.
Repurposing Erectile Dysfunction Drugs Tadalafil and Vardenafil to Increase Bone Mass
Se-Min Kim 1 2, Charit Taneja 3 2, Helena Perez-Pena 4, Vitaly Ryu 3 2, Anisa Gumerova 3 2, Wenliang Li 4, Naseer Ahmad 3 2, Ling-Ling Zhu 3 2, Peng Liu 3 2, Mehr Mathew 3 2, Funda Korkmaz 3 2, Sakshi Gera 3 2, Damini Sant 3 2, Elina Hadelia 3 2, Kseniia Ievleva 3 2 5, Tan-Chun Kuo 3 2, Hirotaka Miyashita 3 2, Li Liu 6 7, Irina Tourkova 6 7, Sarah Stanley 2, Daria Lizneva 3 2, Jameel Iqbal 3 2, Li Sun 3 2, Ronald Tamler 2, Harry C Blair 6 7, Maria I New 1 8, Shozeb Haider 4, Tony Yuen 3 2, Mone Zaidi 3 2
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PMID: 32513693 DOI: 10.1073/pnas.2000950117
Abstract
We report that two widely-used drugs for erectile dysfunction, tadalafil and vardenafil, trigger bone gain in mice through a combination of anabolic and antiresorptive actions on the skeleton. Both drugs were found to enhance osteoblastic bone formation in vivo using a unique gene footprint and to inhibit osteoclast formation. The target enzyme, phosphodiesterase 5A (PDE5A), was found to be expressed in mouse and human bone as well as in specific brain regions, namely the locus coeruleus, raphe pallidus, and paraventricular nucleus of the hypothalamus. Localization of PDE5A in sympathetic neurons was confirmed by coimmunolabeling with dopamine β-hydroxylase, as well as by retrograde bone-brain tracing using a sympathetic nerve-specific pseudorabies virus, PRV152. Both drugs elicited an antianabolic sympathetic imprint in osteoblasts, but with net bone gain. Unlike in humans, in whom vardenafil is more potent than tadalafil, the relative potencies were reversed with respect to their osteoprotective actions in mice. Structural modeling revealed a higher binding energy of tadalafil to mouse PDE5A compared with vardenafil, due to steric clashes of vardenafil with a single methionine residue at position 806 in mouse PDE5A. Collectively, our findings suggest that a balance between peripheral and central actions of PDE5A inhibitors on bone formation together with their antiresorptive actions specify the osteoprotective action of PDE5A blockade.

Keywords: PDE5 inhibitor; computational modeling; cyclic GMP; osteoporosis; resorption.

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The authors declare no competing interest.

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19
Proc Natl Acad Sci U S A
. 2020 Jun 8;202003277. doi: 10.1073/pnas.2003277117. Online ahead of print.
cGMP via PKG Activates 26S Proteasomes and Enhances Degradation of Proteins, Including Ones That Cause Neurodegenerative Diseases
Jordan J S VerPlank 1, Sylwia D Tyrkalska 2 3 4, Angeleen Fleming 2 3 4, David C Rubinsztein 2 3, Alfred L Goldberg 5
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PMID: 32513741 DOI: 10.1073/pnas.2003277117
Free article
Abstract
Because raising cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, we investigated whether agents that raise cGMP may also regulate protein degradation. Treating various cell lines with inhibitors of phosphodiesterase 5 or stimulators of soluble guanylyl cyclase rapidly enhanced multiple proteasome activities and cellular levels of ubiquitinated proteins by activating protein kinase G (PKG). PKG stimulated purified 26S proteasomes by phosphorylating a different 26S component than is modified by protein kinase A. In cells and cell extracts, raising cGMP also enhanced within minutes ubiquitin conjugation to cell proteins. Raising cGMP, like raising cAMP, stimulated the degradation of short-lived cell proteins, but unlike cAMP, also markedly increased proteasomal degradation of long-lived proteins (the bulk of cell proteins) without affecting lysosomal proteolysis. We also tested if raising cGMP, like cAMP, can promote the degradation of mutant proteins that cause neurodegenerative diseases. Treating zebrafish models of tauopathies or Huntington's disease with a PDE5 inhibitor reduced the levels of the mutant huntingtin and tau proteins, cell death, and the resulting morphological abnormalities. Thus, PKG rapidly activates cytosolic proteasomes, protein ubiquitination, and overall protein degradation, and agents that raise cGMP may help combat the progression of neurodegenerative diseases.

Keywords: cGMP; proteasome phosphorylation; protein degradation; protein kinase G.

Copyright © 2020 the Author(s). Published by PNAS.

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The authors declare no competing interest.

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20
Proc Natl Acad Sci U S A
. 2020 Jun 8;202007085. doi: 10.1073/pnas.2007085117. Online ahead of print.
Recognition of Nonproline N-terminal Residues by the Pro/N-degron Pathway
Cheng Dong 1, Shun-Jia Chen 2, Artem Melnykov 3, Sara Weirich 4, Kelly Sun 5, Albert Jeltsch 4, Alexander Varshavsky 6, Jinrong Min 7 8
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PMID: 32513738 DOI: 10.1073/pnas.2007085117
Abstract
Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a K d of 16 μM, whereas the otherwise identical Nt-Pro-bearing sequence PGLW binds to GID4 more tightly, with a K d of 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW-bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.

Keywords: GID; GID4; degradation; degron; ubiquitin.

Conflict of interest statement
The authors declare no competing interest.

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21
Proc Natl Acad Sci U S A
. 2020 Jun 8;201909599. doi: 10.1073/pnas.1909599117. Online ahead of print.
Consistent Scaling of Inbreeding Depression in Space and Time in a House Sparrow Metapopulation
Alina K Niskanen 1 2, Anna M Billing 3, Håkon Holand 3, Ingerid J Hagen 3 4, Yimen G Araya-Ajoy 3, Arild Husby 3 5, Bernt Rønning 3, Ane Marlene Myhre 3, Peter Sjolte Ranke 3, Thomas Kvalnes 3 5, Henrik Pärn 3, Thor Harald Ringsby 3, Sigbjørn Lien 6, Bernt-Erik Sæther 3, Stefanie Muff 7, Henrik Jensen 3
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PMID: 32513746 DOI: 10.1073/pnas.1909599117
Abstract
Inbreeding may increase the extinction risk of small populations. Yet, studies using modern genomic tools to investigate inbreeding depression in nature have been limited to single populations, and little is known about the dynamics of inbreeding depression in subdivided populations over time. Natural populations often experience different environmental conditions and differ in demographic history and genetic composition, characteristics that can affect the severity of inbreeding depression. We utilized extensive long-term data on more than 3,100 individuals from eight islands in an insular house sparrow metapopulation to examine the generality of inbreeding effects. Using genomic estimates of realized inbreeding, we discovered that inbred individuals had lower survival probabilities and produced fewer recruiting offspring than noninbred individuals. Inbreeding depression, measured as the decline in fitness-related traits per unit inbreeding, did not vary appreciably among populations or with time. As a consequence, populations with more resident inbreeding (due to their demographic history) paid a higher total fitness cost, evidenced by a larger variance in fitness explained by inbreeding within these populations. Our results are in contrast to the idea that effects of inbreeding generally depend on ecological factors and genetic differences among populations, and expand the understanding of inbreeding depression in natural subdivided populations.

Keywords: SNP pedigree; house sparrow; inbreeding depression; individual fitness; metapopulation.

Conflict of interest statement
The authors declare no competing interest.

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22
Proc Natl Acad Sci U S A
. 2020 Jun 8;202000810. doi: 10.1073/pnas.2000810117. Online ahead of print.
A Hypoxia-Induced Rab Pathway Regulates Embryo Implantation by Controlled Trafficking of Secretory Granules
Arpita Bhurke 1, Athilakshmi Kannan 1, Alison Neff 2, Qiuyan Ma 2, Mary J Laws 1, Robert N Taylor 3, Milan K Bagchi 4, Indrani C Bagchi 5
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PMID: 32513733 DOI: 10.1073/pnas.2000810117
Abstract
Implantation is initiated when an embryo attaches to the uterine luminal epithelium and subsequently penetrates into the underlying stroma to firmly embed in the endometrium. These events are followed by the formation of an extensive vascular network in the stroma that supports embryonic growth and ensures successful implantation. Interestingly, in many mammalian species, these processes of early pregnancy occur in a hypoxic environment. However, the mechanisms underlying maternal adaptation to hypoxia during early pregnancy remain unclear. In this study, using a knockout mouse model, we show that the transcription factor hypoxia-inducible factor 2 alpha (Hif2α), which is induced in subluminal stromal cells at the time of implantation, plays a crucial role during early pregnancy. Indeed, when preimplantation endometrial stromal cells are exposed to hypoxic conditions in vitro, we observed a striking enhancement in HIF2α expression. Further studies revealed that HIF2α regulates the expression of several metabolic and protein trafficking factors, including RAB27B, at the onset of implantation. RAB27B is a member of the Rab family of GTPases that allows controlled release of secretory granules. These granules are involved in trafficking MMP-9 from the stroma to the epithelium to promote luminal epithelial remodeling during embryo invasion. As pregnancy progresses, the HIF2α-RAB27B pathway additionally mediates crosstalk between stromal and endothelial cells via VEGF granules, developing the vascular network critical for establishing pregnancy. Collectively, our study provides insights into the intercellular communication mechanisms that operate during adaptation to hypoxia, which is essential for embryo implantation and establishment of pregnancy.

Keywords: RAB; endometrium; granules; hypoxia; implantation.

Conflict of interest statement
The authors declare no competing interest.

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23
Proc Natl Acad Sci U S A
. 2020 Jun 8;201916673. doi: 10.1073/pnas.1916673117. Online ahead of print.
Massive Formation of Early Diagenetic Dolomite in the Ediacaran Ocean: Constraints on the "Dolomite Problem"
Biao Chang 1 2, Chao Li 3, Deng Liu 1, Ian Foster 4, Aradhna Tripati 4 5 6, Max K Lloyd 7, Ingrid Maradiaga 5 6, Genming Luo 1, Zhihui An 8, Zhenbing She 1, Shucheng Xie 1, Jinnan Tong 1, Junhua Huang 9, Thomas J Algeo 1 2 10, Timothy W Lyons 11, Adrian Immenhauser 12
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PMID: 32513736 DOI: 10.1073/pnas.1916673117
Abstract
Paleozoic and Precambrian sedimentary successions frequently contain massive dolomicrite [CaMg(CO3)2] units despite kinetic inhibitions to nucleation and precipitation of dolomite at Earth surface temperatures (<60 °C). This paradoxical observation is known as the "dolomite problem." Accordingly, the genesis of these dolostones is usually attributed to burial-hydrothermal dolomitization of primary limestones (CaCO3) at temperatures of >100 °C, thus raising doubt about the validity of these deposits as archives of Earth surface environments. We present a high-resolution, >63-My-long clumped-isotope temperature (TΔ47) record of shallow-marine dolomicrites from two drillcores of the Ediacaran (635 to 541 Ma) Doushantuo Formation in South China. Our T∆47 record indicates that a majority (87%) of these dolostones formed at temperatures of <100 °C. When considering the regional thermal history, modeling of the influence of solid-state reordering on our TΔ47 record further suggests that most of the studied dolostones formed at temperatures of <60 °C, providing direct evidence of a low-temperature origin of these dolostones. Furthermore, calculated δ18O values of diagenetic fluids, rare earth element plus yttrium compositions, and petrographic observations of these dolostones are consistent with an early diagenetic origin in a rock-buffered environment. We thus propose that a precursor precipitate from seawater was subsequently dolomitized during early diagenesis in a near-surface setting to produce the large volume of dolostones in the Doushantuo Formation. Our findings suggest that the preponderance of dolomite in Paleozoic and Precambrian deposits likely reflects oceanic conditions specific to those eras and that dolostones can be faithful recorders of environmental conditions in the early oceans.

Keywords: Doushantuo Formation; carbonate geochemistry; clumped isotope; early diagenesis; early oceans.

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The authors declare no competing interest.

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24
Proc Natl Acad Sci U S A
. 2020 Jun 8;202005353. doi: 10.1073/pnas.2005353117. Online ahead of print.
Hepatocyte-specific TAK1 Deficiency Drives RIPK1 Kinase-Dependent Inflammation to Promote Liver Fibrosis and Hepatocellular Carcinoma
Shuixia Tan 1 2, Jing Zhao 1 2, Ziyu Sun 1 2, Shuangyi Cao 1, Kongyan Niu 1, Yedan Zhong 1, Han Wang 1 2, Linyu Shi 1, Heling Pan 1, Junhao Hu 1, Lihui Qian 1, Nan Liu 3, Junying Yuan 4
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PMID: 32513687 DOI: 10.1073/pnas.2005353117
Abstract
Transforming growth factor β-activated kinase1 (TAK1) encoded by the gene MAP3K7 regulates multiple important downstream effectors involved in immune response, cell death, and carcinogenesis. Hepatocyte-specific deletion of TAK1 in Tak1 ΔHEP mice promotes liver fibrosis and hepatocellular carcinoma (HCC) formation. Here, we report that genetic inactivation of RIPK1 kinase using a kinase dead knockin D138N mutation in Tak1 ΔHEP mice inhibits the expression of liver tumor biomarkers, liver fibrosis, and HCC formation. Inhibition of RIPK1, however, has no or minimum effect on hepatocyte loss and compensatory proliferation, which are the recognized factors important for liver fibrosis and HCC development. Using single-cell RNA sequencing, we discovered that inhibition of RIPK1 strongly suppresses inflammation induced by hepatocyte-specific loss of TAK1. Activation of RIPK1 promotes the transcription of key proinflammatory cytokines, such as CCL2, and CCR2+ macrophage infiltration. Our study demonstrates the role and mechanism of RIPK1 kinase in promoting inflammation, both cell-autonomously and cell-nonautonomously, in the development of liver fibrosis and HCC, independent of cell death, and compensatory proliferation. We suggest the possibility of inhibiting RIPK1 kinase as a therapeutic strategy for reducing liver fibrosis and HCC development by inhibiting inflammation.

Keywords: RIPK1; TAK1; cancer; cell death; inflammation.

Conflict of interest statement
Competing interest statement: J.Y. is a consultant for Denali Therapeutics.

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25
Proc Natl Acad Sci U S A
. 2020 Jun 8;202000317. doi: 10.1073/pnas.2000317117. Online ahead of print.
Deep Rotating Convection Generates the Polar Hexagon on Saturn
Rakesh K Yadav 1, Jeremy Bloxham 2
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PMID: 32513703 DOI: 10.1073/pnas.2000317117
Abstract
Numerous land- and space-based observations have established that Saturn has a persistent hexagonal flow pattern near its north pole. While observations abound, the physics behind its formation is still uncertain. Although several phenomenological models have been able to reproduce this feature, a self-consistent model for how such a large-scale polygonal jet forms in the highly turbulent atmosphere of Saturn is lacking. Here, we present a three-dimensional (3D) fully nonlinear anelastic simulation of deep thermal convection in the outer layers of gas giant planets that spontaneously generates giant polar cyclones, fierce alternating zonal flows, and a high-latitude eastward jet with a polygonal pattern. The analysis of the simulation suggests that self-organized turbulence in the form of giant vortices pinches the eastward jet, forming polygonal shapes. We argue that a similar mechanism is responsible for exciting Saturn's hexagonal flow pattern.

Keywords: planetary atmospheres; rotating convection; turbulence.

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The authors declare no competing interest.

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26
Proc Natl Acad Sci U S A
. 2020 Jun 8;202000681. doi: 10.1073/pnas.2000681117. Online ahead of print.
Emergence of Scale-Free Smectic Rivers and Critical Depinning in Emulsions Driven Through Disorder
Marine Le Blay 1 2, Mokhtar Adda-Bedia 1, Denis Bartolo 3
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PMID: 32513726 DOI: 10.1073/pnas.2000681117
Abstract
During the past 60 min, oil companies have extracted 6 trillion liters of oil from the ground, thereby giving a striking illustration of the impact of multiphase flows on the world economy. From a fundamental perspective, we largely understand the dynamics of interfaces separating immiscible fluids driven through heterogeneous environments. In stark contrast, the basic mechanisms ruling the transport of fragmented fluids, such as foams and emulsions, remain elusive with studies mostly limited to isolated droplets and bubbles. Here, we demonstrate that the mobilization of emulsion driven through model disordered media is a critical plastic depinning transition. To elucidate this collective dynamics, we track the trajectories of hundreds of thousands of microfluidic droplets advected through random lattices of pinning sites. Their dynamics reveals that macroscopic mobilization only requires the coordinated motion of small groups of particles and does not involve any large-scale avalanches. Criticality arises from the interplay between contact and hydrodynamic interaction, which channel seemingly erratic depinning events along smectic river networks correlated over system spanning scales. Beyond the specifics of emulsion transport, we close our article discussing the similarities and profound differences with the plastic depinning transitions of driven flux lines in high-T c superconductors, charged colloids, and grain transport in eroded sand beds.

Keywords: dynamical phase transition; heterogeneous media; plastic depinning; transport.

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The authors declare no competing interest.

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27
Proc Natl Acad Sci U S A
. 2020 Jun 8;201918163. doi: 10.1073/pnas.1918163117. Online ahead of print.
Myeloid ALX/FPR2 Regulates Vascularization Following Tissue Injury
Brian E Sansbury 1, Xiaofeng Li 1, Blenda Wong 1, Andreas Patsalos 2 3, Nikolas Giannakis 4, Michael J Zhang 5, Laszlo Nagy 2 3 4, Matthew Spite 6
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PMID: 32513697 DOI: 10.1073/pnas.1918163117
Abstract
Ischemic injury initiates a sterile inflammatory response that ultimately participates in the repair and recovery of tissue perfusion. Macrophages are required for perfusion recovery during ischemia, in part because they produce growth factors that aid in vascular remodeling. The input signals governing this pro-revascularization phenotype remain of interest. Here we found that hindlimb ischemia increases levels of resolvin D1 (RvD1), an inflammation-resolving lipid mediator that targets macrophages via its receptor, ALX/FPR2. Exogenous RvD1 enhances perfusion recovery during ischemia, and mice deficient in Alx/Fpr2 have an endogenous defect in this process. Mechanistically, RNA sequencing revealed that RvD1 induces a transcriptional program in macrophages characteristic of a pro-revascularization phenotype. Vascularization of ischemic skeletal muscle, as well as cutaneous wounds, is impaired in mice with myeloid-specific deficiency of Alx/Fpr2, and this is associated with altered expression of pro-revascularization genes in skeletal muscle and macrophages isolated from skeletal muscle. Collectively, these results uncover a role of ALX/FPR2 in revascularization that may be amenable to therapeutic targeting in diseases associated with altered tissue perfusion and repair.

Keywords: ischemia; macrophages; resolvins.

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The authors declare no competing interest.

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28
Proc Natl Acad Sci U S A
. 2020 Jun 8;202000497. doi: 10.1073/pnas.2000497117. Online ahead of print.
The Legionella Kinase LegK7 Exploits the Hippo Pathway Scaffold Protein MOB1A for Allostery and Substrate Phosphorylation
Pei-Chung Lee 1 2, Ksenia Beyrakhova 3, Caishuang Xu 3, Michal T Boniecki 3, Mitchell H Lee 1, Chisom J Onu 2, Andrey M Grishin 3, Matthias P Machner 4, Miroslaw Cygler 5
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PMID: 32513747 DOI: 10.1073/pnas.2000497117
Abstract
During infection, the bacterial pathogen Legionella pneumophila manipulates a variety of host cell signaling pathways, including the Hippo pathway which controls cell proliferation and differentiation in eukaryotes. Our previous studies revealed that L. pneumophila encodes the effector kinase LegK7 which phosphorylates MOB1A, a highly conserved scaffold protein of the Hippo pathway. Here, we show that MOB1A, in addition to being a substrate of LegK7, also functions as an allosteric activator of its kinase activity. A crystallographic analysis of the LegK7-MOB1A complex revealed that the N-terminal half of LegK7 is structurally similar to eukaryotic protein kinases, and that MOB1A directly binds to the LegK7 kinase domain. Substitution of interface residues critical for complex formation abrogated allosteric activation of LegK7 both in vitro and within cells and diminished MOB1A phosphorylation. Importantly, the N-terminal extension (NTE) of MOB1A not only regulated complex formation with LegK7 but also served as a docking site for downstream substrates such as the transcriptional coregulator YAP1. Deletion of the NTE from MOB1A or addition of NTE peptides as binding competitors attenuated YAP1 recruitment to and phosphorylation by LegK7. By providing mechanistic insight into the formation and regulation of the LegK7-MOB1A complex, our study unravels a sophisticated molecular mimicry strategy that is used by L. pneumophila to take control of the host cell Hippo pathway.

Keywords: MST1/2; crystal structure; microbial pathogen; translocated effector; type IV secretion system.

Conflict of interest statement
The authors declare no competing interest.

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29
Proc Natl Acad Sci U S A
. 2020 Jun 8;201911439. doi: 10.1073/pnas.1911439117. Online ahead of print.
Using Gross Ecosystem Product (GEP) to Value Nature in Decision Making
Zhiyun Ouyang 1 2, Changsu Song 1 2, Hua Zheng 1 2, Stephen Polasky 3 4, Yi Xiao 1 2, Ian J Bateman 5, Jianguo Liu 6 7, Mary Ruckelshaus 8, Faqi Shi 9, Yang Xiao 1, Weihua Xu 1 2, Ziying Zou 2 10, Gretchen C Daily 8 11
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PMID: 32513694 DOI: 10.1073/pnas.1911439117
Free article
Abstract
Gross domestic product (GDP) summarizes a vast amount of economic information in a single monetary metric that is widely used by decision makers around the world. However, GDP fails to capture fully the contributions of nature to economic activity and human well-being. To address this critical omission, we develop a measure of gross ecosystem product (GEP) that summarizes the value of ecosystem services in a single monetary metric. We illustrate the measurement of GEP through an application to the Chinese province of Qinghai, showing that the approach is tractable using available data. Known as the "water tower of Asia," Qinghai is the source of the Mekong, Yangtze, and Yellow Rivers, and indeed, we find that water-related ecosystem services make up nearly two-thirds of the value of GEP for Qinghai. Importantly most of these benefits accrue downstream. In Qinghai, GEP was greater than GDP in 2000 and three-fourths as large as GDP in 2015 as its market economy grew. Large-scale investment in restoration resulted in improvements in the flows of ecosystem services measured in GEP (127.5%) over this period. Going forward, China is using GEP in decision making in multiple ways, as part of a transformation to inclusive, green growth. This includes investing in conservation of ecosystem assets to secure provision of ecosystem services through transregional compensation payments.

Keywords: GDP; ecosystem services; environmental–economic accounting; gross ecosystem product; natural capital.

Copyright © 2020 the Author(s). Published by PNAS.

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The authors declare no competing interest.

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30
Proc Natl Acad Sci U S A
. 2020 Jun 8;202002580. doi: 10.1073/pnas.2002580117. Online ahead of print.
Global Impact of Atmospheric Arsenic on Health Risk: 2005 to 2015
Lei Zhang 1, Yang Gao 2 3, Shiliang Wu 4, Shaoqing Zhang 5 6 7, Kirk R Smith 8 9, Xiaohong Yao 1 3, Huiwang Gao 1 3
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PMID: 32513708 DOI: 10.1073/pnas.2002580117
Abstract
Arsenic is a toxic pollutant commonly found in the environment. Most of the previous studies on arsenic pollution have primarily focused on arsenic contamination in groundwater. In this study, we examine the impact on human health from atmospheric arsenic on the global scale. We first develop an improved global atmospheric arsenic emission inventory and connect it to a global model (Goddard Earth Observing System [GEOS]-Chem). Model evaluation using observational data from a variety of sources shows the model successfully reproduces the spatial distribution of atmospheric arsenic around the world. We found that for 2005, the highest airborne arsenic concentrations were found over Chile and eastern China, with mean values of 8.34 and 5.63 ng/m3, respectively. By 2015, the average atmospheric arsenic concentration in India (4.57 ng/m3) surpassed that in eastern China (4.38 ng/m3) due to the fast increase in coal burning in India. Our calculation shows that China has the largest population affected by cancer risk due to atmospheric arsenic inhalation in 2005, which is again surpassed by India in 2015. Based on potential exceedance of health-based limits, we find that the combined effect by including both atmospheric and groundwater arsenic may significantly enhance the risks, due to carcinogenic and noncarcinogenic effects. Therefore, this study clearly implies the necessity in accounting for both atmospheric and groundwater arsenic in future management.

Keywords: GEOS-Chem; atmospheric arsenic; cancer risk; noncarcinogenic effect.

Conflict of interest statement
The authors declare no competing interest.

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31
Proc Natl Acad Sci U S A
. 2020 Jun 8;201909683. doi: 10.1073/pnas.1909683117. Online ahead of print.
Cotranslational Folding Cooperativity of Contiguous Domains of α-Spectrin
Grant Kemp 1, Ola B Nilsson 1 2, Pengfei Tian 3, Robert B Best 4, Gunnar von Heijne 5 6
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PMID: 32513720 DOI: 10.1073/pnas.1909683117
Abstract
Proteins synthesized in the cell can begin to fold during translation before the entire polypeptide has been produced, which may be particularly relevant to the folding of multidomain proteins. Here, we study the cotranslational folding of adjacent domains from the cytoskeletal protein α-spectrin using force profile analysis (FPA). Specifically, we investigate how the cotranslational folding behavior of the R15 and R16 domains are affected by their neighboring R14 and R16, and R15 and R17 domains, respectively. Our results show that the domains impact each other's folding in distinct ways that may be important for the efficient assembly of α-spectrin, and may reduce its dependence on chaperones. Furthermore, we directly relate the experimentally observed yield of full-length protein in the FPA assay to the force exerted by the folding protein in piconewtons. By combining pulse-chase experiments to measure the rate at which the arrested protein is converted into full-length protein with a Bell model of force-induced rupture, we estimate that the R16 domain exerts a maximal force on the nascent chain of ∼15 pN during cotranslational folding.

Keywords: SecM; molecular dynamics; protein folding; spectrin; translational arrest peptide.

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The authors declare no competing interest.

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32
Proc Natl Acad Sci U S A
. 2020 Jun 8;202001806. doi: 10.1073/pnas.2001806117. Online ahead of print.
Structural Basis for Plant Lutein Biosynthesis From α-Carotene
Guoqi Niu 1 2, Qi Guo 1 3, Jia Wang 1 3, Shun Zhao 1, Yikun He 4, Lin Liu 5
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PMID: 32513704 DOI: 10.1073/pnas.2001806117
Abstract
Two cytochrome P450 enzymes, CYP97A3 and CYP97C1, catalyze hydroxylations of the β- and ε-rings of α-carotene to produce lutein. Chirality is introduced at the C-3 atom of both rings, and the reactions are both pro-3R-stereospecific. We determined the crystal structures of CYP97A3 in substrate-free and complex forms with a nonnatural substrate and the structure of CYP97C1 in a detergent-bound form. The structures of CYP97A3 in different states show the substrate channel and the structure of CYP97C1 bound with octylthioglucoside confirms the binding site for the carotenoid substrate. Biochemical assays confirm that the ferredoxin-NADP+ reductase (FNR)-ferredoxin pair is used as the redox partner. Details of the pro-3R stereospecificity are revealed in the retinal-bound CYP97A3 structure. Further analysis indicates that the CYP97B clan bears similarity to the β-ring-specific CYP97A clan. Overall, our research describes the molecular basis for the last steps of lutein biosynthesis.

Keywords: P450; chirality; hydroxylation; stereospecificity; xanthophyll.

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The authors declare no competing interest.

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33
Proc Natl Acad Sci U S A
. 2020 Jun 8;201920165. doi: 10.1073/pnas.1920165117. Online ahead of print.
Topoisomerase I-driven Repair of UV-induced Damage in NER-deficient Cells
Liton Kumar Saha 1 2, Mitsuo Wakasugi 3, Salma Akter 1, Rajendra Prasad 4, Samuel H Wilson 4, Naoto Shimizu 1, Hiroyuki Sasanuma 1, Shar-Yin Naomi Huang 2, Keli Agama 2, Yves Pommier 2, Tsukasa Matsunaga 3, Kouji Hirota 5, Shigenori Iwai 6, Yuka Nakazawa 7, Tomoo Ogi 7, Shunichi Takeda 8
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PMID: 32513688 DOI: 10.1073/pnas.1920165117
Abstract
Nucleotide excision repair (NER) removes helix-destabilizing adducts including ultraviolet (UV) lesions, cyclobutane pyrimidine dimers (CPDs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). In comparison with CPDs, 6-4PPs have greater cytotoxicity and more strongly destabilizing properties of the DNA helix. It is generally believed that NER is the only DNA repair pathway that removes the UV lesions as evidenced by the previous data since no repair of UV lesions was detected in NER-deficient skin fibroblasts. Topoisomerase I (TOP1) constantly creates transient single-strand breaks (SSBs) releasing the torsional stress in genomic duplex DNA. Stalled TOP1-SSB complexes can form near DNA lesions including abasic sites and ribonucleotides embedded in chromosomal DNA. Here we show that base excision repair (BER) increases cellular tolerance to UV independently of NER in cancer cells. UV lesions irreversibly trap stable TOP1-SSB complexes near the UV damage in NER-deficient cells, and the resulting SSBs activate BER. Biochemical experiments show that 6-4PPs efficiently induce stable TOP1-SSB complexes, and the long-patch repair synthesis of BER removes 6-4PPs downstream of the SSB. Furthermore, NER-deficient cancer cell lines remove 6-4PPs within 24 h, but not CPDs, and the removal correlates with TOP1 expression. NER-deficient skin fibroblasts weakly express TOP1 and show no detectable repair of 6-4PPs. Remarkably, the ectopic expression of TOP1 in these fibroblasts led them to completely repair 6-4PPs within 24 h. In conclusion, we reveal a DNA repair pathway initiated by TOP1, which significantly contributes to cellular tolerance to UV-induced lesions particularly in malignant cancer cells overexpressing TOP1.

Keywords: 6–4PPs; UV damage; base excision repair; topoisomerase I.

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The authors declare no competing interest.

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34
Proc Natl Acad Sci U S A
. 2020 Jun 8;201918596. doi: 10.1073/pnas.1918596117. Online ahead of print.
RIG-I Regulates Myeloid Differentiation by Promoting TRIM25-mediated ISGylation
Song-Fang Wu 1 2, Li Xia 1, Xiao-Dong Shi 1, Yu-Jun Dai 1, Wei-Na Zhang 1, Jun-Mei Zhao 1, Wu Zhang 1, Xiang-Qin Weng 1, Jing Lu 1, Huang-Ying Le 2, Sheng-Ce Tao 2, Jiang Zhu 1, Zhu Chen 3 2, Yue-Ying Wang 3, Saijuan Chen 3 2
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PMID: 32513696 DOI: 10.1073/pnas.1918596117
Abstract
Retinoic acid-inducible gene I (RIG-I) is up-regulated during granulocytic differentiation of acute promyelocytic leukemia (APL) cells induced by all-trans retinoic acid (ATRA). It has been reported that RIG-I recognizes virus-specific 5'-ppp-double-stranded RNA (dsRNA) and activates the type I interferons signaling pathways in innate immunity. However, the functions of RIG-I in hematopoiesis remain unclear, especially regarding its possible interaction with endogenous RNAs and the associated pathways that could contribute to the cellular differentiation and maturation. Herein, we identified a number of RIG-I-binding endogenous RNAs in APL cells following ATRA treatment, including the tripartite motif-containing protein 25 (TRIM25) messenger RNA (mRNA). TRIM25 encodes the protein known as an E3 ligase for ubiquitin/interferon (IFN)-induced 15-kDa protein (ISG15) that is involved in RIG-I-mediated antiviral signaling. We show that RIG-I could bind TRIM25 mRNA via its helicase domain and C-terminal regulatory domain, enhancing the stability of TRIM25 transcripts. RIG-I could increase the transcriptional expression of TRIM25 by caspase recruitment domain (CARD) domain through an IFN-stimulated response element. In addition, RIG-I activated other key genes in the ISGylation pathway by activating signal transducer and activator of transcription 1 (STAT1), including the modifier ISG15 and several enzymes responsible for the conjugation of ISG15 to protein substrates. RIG-I cooperated with STAT1/2 and interferon regulatory factor 1 (IRF1) to promote the activation of the ISGylation pathway. The integrity of ISGylation in ATRA or RIG-I-induced cell differentiation was essential given that knockdown of TRIM25 or ISG15 resulted in significant inhibition of this process. Our results provide insight into the role of the RIG-I-TRIM25-ISGylation axis in myeloid differentiation.

Keywords: ISGylation; RIG-I; TRIM25; acute promyelocytic leukemia (APL); myeloid differentiation.

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The authors declare no competing interest.

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35
Proc Natl Acad Sci U S A
. 2020 Jun 8;201913469. doi: 10.1073/pnas.1913469117. Online ahead of print.
O-GlcNAcylation on LATS2 Disrupts the Hippo Pathway by Inhibiting Its Activity
Eunah Kim 1 2, Jeong Gu Kang 3, Min Jueng Kang 4, Jae Hyung Park 5, Yeon Jung Kim 1 2, Tae Hyun Kweon 1 2, Han-Woong Lee 5, Eek-Hoon Jho 1 6, Yong-Ho Lee 1 2, Seung-Il Kim 7, Eugene C Yi 1 4, Hyun Woo Park 5, Won Ho Yang 1 8, Jin Won Cho 9 2 8
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PMID: 32513743 DOI: 10.1073/pnas.1913469117
Free article
Abstract
The Hippo pathway controls organ size and tissue homeostasis by regulating cell proliferation and apoptosis. The LATS-mediated negative feedback loop prevents excessive activation of the effectors YAP/TAZ, maintaining homeostasis of the Hippo pathway. YAP and TAZ are hyperactivated in various cancer cells which lead to tumor growth. Aberrantly increased O-GlcNAcylation has recently emerged as a cause of hyperactivation of YAP in cancer cells. However, the mechanism, which induces hyperactivation of TAZ and blocks LATS-mediated negative feedback, remains to be elucidated in cancer cells. This study found that in breast cancer cells, abnormally increased O-GlcNAcylation hyperactivates YAP/TAZ and inhibits LATS2, a direct negative regulator of YAP/TAZ. LATS2 is one of the newly identified O-GlcNAcylated components in the MST-LATS kinase cascade. Here, we found that O-GlcNAcylation at LATS2 Thr436 interrupted its interaction with the MOB1 adaptor protein, which connects MST to LATS2, leading to activation of YAP/TAZ by suppressing LATS2 kinase activity. LATS2 is a core component in the LATS-mediated negative feedback loop. Thus, this study suggests that LATS2 O-GlcNAcylation is deeply involved in tumor growth by playing a critical role in dysregulation of the Hippo pathway in cancer cells.

Keywords: Hippo pathway; LATS2; MOB1; O-GlcNAcylation; cancer.

Copyright © 2020 the Author(s). Published by PNAS.

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The authors declare no competing interest.

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36
Proc Natl Acad Sci U S A
. 2020 Jun 8;201922525. doi: 10.1073/pnas.1922525117. Online ahead of print.
CD5 Dynamically Calibrates Basal NF-κB Signaling in T Cells During Thymic Development and Peripheral Activation
Courtney A Matson 1, Seeyoung Choi 2, Ferenc Livak 3, Bin Zhao 2, Apratim Mitra 4, Paul E Love 2, Nevil J Singh 5
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PMID: 32513716 DOI: 10.1073/pnas.1922525117
Abstract
Immature T cells undergo a process of positive selection in the thymus when their new T cell receptor (TCR) engages and signals in response to self-peptides. As the T cell matures, a slew of negative regulatory molecules, including the inhibitory surface glycoprotein CD5, are up-regulated in proportion to the strength of the self-peptide signal. Together these regulators dampen TCR-proximal signaling and help avoid any subsequent peripheral activation of T cells by self-peptides. Paradoxically, antigen-specific T cells initially expressing more CD5 (CD5hi) have been found to better persist as effector/memory cells after a peripheral challenge. The molecular mechanisms underlying such a duality in CD5 function is not clear. We found that CD5 alters the basal activity of the NF-κB signaling in resting peripheral T cells. When CD5 was conditionally ablated, T cells were unable to maintain higher expression of the cytoplasmic NF-κB inhibitor IκBα. Consistent with this, resting CD5hi T cells expressed more of the NF-κB p65 protein than CD5lo cells, without significant increases in transcript levels, in the absence of TCR signals. This posttranslationally stabilized cellular NF-κB depot potentially confers a survival advantage to CD5hi T cells over CD5lo ones. Taken together, these data suggest a two-step model whereby the strength of self-peptide-induced TCR signal lead to the up-regulation of CD5, which subsequently maintains a proportional reserve of NF-κB in peripheral T cells poised for responding to agonistic antigen-driven T cell activation.

Keywords: CD5; T cell activation; TCR signaling.

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The authors declare no competing interest.

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37
Proc Natl Acad Sci U S A
. 2020 Jun 8;201922903. doi: 10.1073/pnas.1922903117. Online ahead of print.
Employing NaChBac for cryo-EM Analysis of Toxin Action on Voltage-Gated Na + Channels in Nanodisc
Shuai Gao 1, William C Valinsky 2, Nguyen Cam On 1, Patrick R Houlihan 2, Qian Qu 3, Lei Liu 3, Xiaojing Pan 4, David E Clapham 2, Nieng Yan 5
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PMID: 32513729 DOI: 10.1073/pnas.1922903117
Abstract
NaChBac, the first bacterial voltage-gated Na+ (Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Nav channels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the single particle electron cryomicroscopy (cryo-EM) analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Nav channels in lipid bilayers. To study GMTs in mammalian Nav channels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 replaced the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV-bound human Nav1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from ∼6 to ∼4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Nav channels in a membrane environment.

Keywords: NaChBac; Nav channels; electron cryomicroscopy (cryo-EM); gating modifier toxins; nanodisc.

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The authors declare no competing interest.

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38
Proc Natl Acad Sci U S A
. 2020 Jun 8;201918272. doi: 10.1073/pnas.1918272117. Online ahead of print.
The Appearance and Duration of the Jehol Biota: Constraint From SIMS U-Pb Zircon Dating for the Huajiying Formation in Northern China
Saihong Yang 1 2, Huaiyu He 3 2 4, Fan Jin 5, Fucheng Zhang 6, Yuanbao Wu 7, Zhiqiang Yu 1 2 4, Qiuli Li 1 2 4, Min Wang 5 8, Jingmai K O'Connor 5 8, Chenglong Deng 1 2 4, Rixiang Zhu 1 2 4, Zhonghe Zhou 9 8
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PMID: 32513701 DOI: 10.1073/pnas.1918272117
Free article
Abstract
The Lower Cretaceous Huajiying Formation of the Sichakou Basin in northern Hebei Province, northern China contains key vertebrate taxa of the early Jehol Biota, e.g., Protopteryx fengningensis, Archaeornithura meemannae, Peipiaosteus fengningensis, and Eoconfuciusornis zhengi This formation arguably documents the second-oldest bird-bearing horizon, producing the oldest fossil records of the two major Mesozoic avian groups Enantiornithes and Ornithuromorpha. Hence, precisely determining the depositional ages of the Huajiying Formation would advance our understanding of the evolutionary history of the Jehol Biota. Here we present secondary ion mass spectrometry (SIMS) U-Pb zircon analysis results of eight interbedded tuff/tuffaceous sandstone samples from the Huajiying Formation. Our findings, combined with previous radiometric dates, suggest that the oldest enantiornithine and ornithuromorph birds in the Jehol Biota are ∼129-131 Ma, and that the Jehol Biota most likely first appeared at ∼135 Ma. This expands the biota's temporal distribution from late Valanginian to middle Aptian with a time span of about 15 My.

Keywords: Cretaceous; Jehol Biota; U-Pb geochronology.

Copyright © 2020 the Author(s). Published by PNAS.

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39
Proc Natl Acad Sci U S A
. 2020 Jun 8;202002981. doi: 10.1073/pnas.2002981117. Online ahead of print.
Mirrored Brain Organization: Statistical Anomaly or Reversal of Hemispheric Functional Segregation Bias?
Robin Gerrits 1, Helena Verhelst 2, Guy Vingerhoets 2 3
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PMID: 32513702 DOI: 10.1073/pnas.2002981117
Abstract
Humans demonstrate a prototypical hemispheric functional segregation pattern, with language and praxis lateralizing to the left hemisphere and spatial attention, face recognition, and emotional prosody to the right hemisphere. In this study, we used fMRI to determine laterality for all five functions in each participant. Crucially, we recruited a sample of left-handers preselected for atypical (right) language dominance (n = 24), which allowed us to characterize hemispheric asymmetry of the other functions and compare their functional segregation pattern with that of left-handers showing typical language dominance (n = 39). Our results revealed that most participants with left language dominance display the prototypical pattern of functional hemispheric segregation (44%) or deviate from this pattern in only one function (35%). Similarly, the vast majority of right language dominant participants demonstrated a completely mirrored brain organization (50%) or a reversal for all but one cognitive function (32%). Participants deviating by more than one function from the standard segregation pattern showed poorer cognitive performance, in line with an oft-presumed biological advantage of hemispheric functional segregation.

Keywords: functional brain asymmetry; hemispheric dominance; hemispheric segregation; language dominance; lateralization.

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40
Proc Natl Acad Sci U S A
. 2020 Jun 8;201914670. doi: 10.1073/pnas.1914670117. Online ahead of print.
Single-vesicle Imaging Reveals Lipid-Selective and Stepwise Membrane Disruption by Monomeric α-Synuclein
Jonas K Hannestad 1, Sandra Rocha 2, Björn Agnarsson 1, Vladimir P Zhdanov 1 3, Pernilla Wittung-Stafshede 4, Fredrik Höök 5
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PMID: 32513706 DOI: 10.1073/pnas.1914670117
Free article
Abstract
The interaction of the neuronal protein α-synuclein with lipid membranes appears crucial in the context of Parkinson's disease, but the underlying mechanistic details, including the roles of different lipids in pathogenic protein aggregation and membrane disruption, remain elusive. Here, we used single-vesicle resolution fluorescence and label-free scattering microscopy to investigate the interaction kinetics of monomeric α-synuclein with surface-tethered vesicles composed of different negatively charged lipids. Supported by a theoretical model to account for structural changes in scattering properties of surface-tethered lipid vesicles, the data demonstrate stepwise vesicle disruption and asymmetric membrane deformation upon α-synuclein binding to phosphatidylglycerol vesicles at protein concentrations down to 10 nM (∼100 proteins per vesicle). In contrast, phosphatidylserine vesicles were only marginally affected. These insights into structural consequences of α-synuclein interaction with lipid vesicles highlight the contrasting roles of different anionic lipids, which may be of mechanistic relevance for both normal protein function (e.g., synaptic vesicle binding) and dysfunction (e.g., mitochondrial membrane interaction).

Keywords: lipid vesicle; membrane interaction; single-vesicle scattering; α-synuclein.

Copyright © 2020 the Author(s). Published by PNAS.

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The authors declare no competing interest.

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41
Proc Natl Acad Sci U S A
. 2020 Jun 8;202002924. doi: 10.1073/pnas.2002924117. Online ahead of print.
How a Raindrop Gets Shattered on Biological Surfaces
Seungho Kim 1, Zixuan Wu 1, Ehsan Esmaili 1, Jason J Dombroskie 2, Sunghwan Jung 3
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PMID: 32513723 DOI: 10.1073/pnas.2002924117
Free article
Abstract
Many biological surfaces of animals and plants (e.g., bird feathers, insect wings, plant leaves, etc.) are superhydrophobic with rough surfaces at different length scales. Previous studies have focused on a simple drop-bouncing behavior on biological surfaces with low-speed impacts. However, we observed that an impacting drop at high speeds exhibits more complicated dynamics with unexpected shock-like patterns: Hundreds of shock-like waves are formed on the spreading drop, and the drop is then abruptly fragmented along with multiple nucleating holes. Such drop dynamics result in the rapid retraction of the spreading drop and thereby a more than twofold decrease in contact time. Our results may shed light on potential biological advantages of hypothermia risk reduction for endothermic animals and spore spreading enhancement for fungi via wave-induced drop fragmentation.

Keywords: contact time; drop impact; microstructures; superhydrophobic surfaces.

Copyright © 2020 the Author(s). Published by PNAS.

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The authors declare no competing interest.

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42
Proc Natl Acad Sci U S A
. 2020 Jun 8;201920201. doi: 10.1073/pnas.1920201117. Online ahead of print.
Atypical Protein Kinase C Iota (PKCλ/ι) Ensures Mammalian Development by Establishing the Maternal-Fetal Exchange Interface
Bhaswati Bhattacharya 1, Pratik Home 1 2, Avishek Ganguly 1, Soma Ray 1, Ananya Ghosh 1, Md Rashedul Islam 1, Valerie French 2 3, Courtney Marsh 2 3, Sumedha Gunewardena 4, Hiroaki Okae 5, Takahiro Arima 5, Soumen Paul 6 2 3
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PMID: 32513715 DOI: 10.1073/pnas.1920201117
Free article
Abstract
In utero mammalian development relies on the establishment of the maternal-fetal exchange interface, which ensures transportation of nutrients and gases between the mother and the fetus. This exchange interface is established via development of multinucleated syncytiotrophoblast cells (SynTs) during placentation. In mice, SynTs develop via differentiation of the trophoblast stem cell-like progenitor cells (TSPCs) of the placenta primordium, and in humans, SynTs are developed via differentiation of villous cytotrophoblast (CTB) progenitors. Despite the critical need in pregnancy progression, conserved signaling mechanisms that ensure SynT development are poorly understood. Herein, we show that atypical protein kinase C iota (PKCλ/ι) plays an essential role in establishing the SynT differentiation program in trophoblast progenitors. Loss of PKCλ/ι in the mouse TSPCs abrogates SynT development, leading to embryonic death at approximately embryonic day 9.0 (E9.0). We also show that PKCλ/ι-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. PKCλ/ι is selectively expressed in the first-trimester CTBs of a developing human placenta. Furthermore, loss of PKCλ/ι in CTB-derived human trophoblast stem cells (human TSCs) impairs their SynT differentiation potential both in vitro and after transplantation in immunocompromised mice. Our mechanistic analyses indicate that PKCλ/ι signaling maintains expression of GCM1, GATA2, and PPARγ, which are key transcription factors to instigate SynT differentiation programs in both mouse and human trophoblast progenitors. Our study uncovers a conserved molecular mechanism, in which PKCλ/ι signaling regulates establishment of the maternal-fetal exchange surface by promoting trophoblast progenitor-to-SynT transition during placentation.

Keywords: cytotrophoblast; human trophoblast stem cell; placenta; protein kinase Cλ/ι; syncytiotrophoblast.

Copyright © 2020 the Author(s). Published by PNAS.

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43
Proc Natl Acad Sci U S A
. 2020 Jun 8;202003225. doi: 10.1073/pnas.2003225117. Online ahead of print.
Amorphous Polymer Dynamics and Free Volume Element Size Distributions From Ultrafast IR Spectroscopy
David J Hoffman 1, Sebastian M Fica-Contreras 1, Michael D Fayer 2
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PMID: 32513742 DOI: 10.1073/pnas.2003225117
Abstract
A method for measuring the size and size probability distribution of free volume regions in polymeric materials using ultrafast infrared (IR) polarization-selective pump-probe experiments is presented. Measurements of the ultrafast dynamics of a vibrational probe (the CN stretch of phenyl selenocyanate) in poly(methyl methacrylate) show that the probe dynamics are highly confined. The degree of confinement was found to be both time-dependent and dependent on the vibrational frequency of the probe molecule. The experiments demonstrate that different vibrational frequencies correspond to distinct subensembles of probe molecules that have different dynamic properties determined by their local structural environments. By combining the degree of dynamical confinement with the molecular size of the probe molecule, the free volume element size probability distribution was determined and found to be in good agreement with the best established experimental measure of free volume. The relative probability of a free volume element size is determined by the amplitude of the nitrile absorption spectrum at the frequency of the measurement. The inhomogeneous broadening of the spectrum was linked to the vibrational Stark effect, which permits site selectivity. The observed dynamics at each frequency were then associated with a different size free volume element and distinct local electric field. The multiple timescales observed in the pump-probe experiments were connected to local structural fluctuations of the free volume elements.

Keywords: Stark effect; dynamics; free volume; polymers; ultrafast IR spectroscopy.

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The authors declare no competing interest.

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44
Proc Natl Acad Sci U S A
. 2020 Jun 8;201916787. doi: 10.1073/pnas.1916787117. Online ahead of print.
Functional Ultrasound Imaging of Deep Visual Cortex in Awake Nonhuman Primates
Kévin Blaize 1, Fabrice Arcizet 2, Marc Gesnik 3, Harry Ahnine 4, Ulisse Ferrari 2, Thomas Deffieux 3, Pierre Pouget 4, Frédéric Chavane 5, Mathias Fink 2, José-Alain Sahel 2 6 7, Mickael Tanter 3, Serge Picaud 1
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PMID: 32513717 DOI: 10.1073/pnas.1916787117
Free article
Abstract
Deep regions of the brain are not easily accessible to investigation at the mesoscale level in awake animals or humans. We have recently developed a functional ultrasound (fUS) technique that enables imaging hemodynamic responses to visual tasks. Using fUS imaging on two awake nonhuman primates performing a passive fixation task, we constructed retinotopic maps at depth in the visual cortex (V1, V2, and V3) in the calcarine and lunate sulci. The maps could be acquired in a single-hour session with relatively few presentations of the stimuli. The spatial resolution of the technology is illustrated by mapping patterns similar to ocular dominance (OD) columns within superficial and deep layers of the primary visual cortex. These acquisitions using fUS suggested that OD selectivity is mostly present in layer IV but with extensions into layers II/III and V. This imaging technology provides a new mesoscale approach to the mapping of brain activity at high spatiotemporal resolution in awake subjects within the whole depth of the cortex.

Keywords: brain imaging; functional ultrasound imaging; nonhuman primate; ocular dominance; visual cortex.

Copyright © 2020 the Author(s). Published by PNAS.

Conflict of interest statement
Competing interest statement: T.D. and M.T. are cofounders and T.D., M.T., and S.P. are shareholders of ICONEUS.

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45
Proc Natl Acad Sci U S A
. 2020 Jun 8;202004106. doi: 10.1073/pnas.2004106117. Online ahead of print.
Unveiling Defect-Mediated Carrier Dynamics in Monolayer Semiconductors by Spatiotemporal Microwave Imaging
Zhaodong Chu 1, Chun-Yuan Wang 1, Jiamin Quan 1, Chenhui Zhang 2, Chao Lei 1, Ali Han 2, Xuejian Ma 1, Hao-Ling Tang 2, Dishan Abeysinghe 1, Matthew Staab 1, Xixiang Zhang 2, Allan H MacDonald 1, Vincent Tung 2, Xiaoqin Li 1, Chih-Kang Shih 1, Keji Lai 3
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PMID: 32513713 DOI: 10.1073/pnas.2004106117
Abstract
The optoelectronic properties of atomically thin transition-metal dichalcogenides are strongly correlated with the presence of defects in the materials, which are not necessarily detrimental for certain applications. For instance, defects can lead to an enhanced photoconduction, a complicated process involving charge generation and recombination in the time domain and carrier transport in the spatial domain. Here, we report the simultaneous spatial and temporal photoconductivity imaging in two types of WS2 monolayers by laser-illuminated microwave impedance microscopy. The diffusion length and carrier lifetime were directly extracted from the spatial profile and temporal relaxation of microwave signals, respectively. Time-resolved experiments indicate that the critical process for photoexcited carriers is the escape of holes from trap states, which prolongs the apparent lifetime of mobile electrons in the conduction band. As a result, counterintuitively, the long-lived photoconductivity signal is higher in chemical-vapor deposited (CVD) samples than exfoliated monolayers due to the presence of traps that inhibits recombination. Our work reveals the intrinsic time and length scales of electrical response to photoexcitation in van der Waals materials, which is essential for their applications in optoelectronic devices.

Keywords: charge carriers; defects; laser-illuminated microwave impedance microscopy; spatiotemporal dynamics; transition-metal dichalcogenides.

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The authors declare no competing interest.

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46
Proc Natl Acad Sci U S A
. 2020 Jun 8;202003019. doi: 10.1073/pnas.2003019117. Online ahead of print.
Spatial Confinement of Receptor Activity by Tyrosine Phosphatase During Directional Cell Migration
Zhiwen Zhu 1 2 3 4, Yongping Chai 1 2 3 4, Huifang Hu 3, Wei Li 5, Wen-Jun Li 6, Meng-Qiu Dong 6, Jia-Wei Wu 3, Zhi-Xin Wang 3, Guangshuo Ou 7 2 3 4
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PMID: 32513699 DOI: 10.1073/pnas.2003019117
Abstract
Directional cell migration involves signaling cascades that stimulate actin assembly at the leading edge, and additional pathways must inhibit actin polymerization at the rear. During neuroblast migration in Caenorhabditis elegans, the transmembrane protein MIG-13/Lrp12 acts through the Arp2/3 nucleation-promoting factors WAVE and WASP to guide the anterior migration. Here we show that a tyrosine kinase, SRC-1, directly phosphorylates MIG-13 and promotes its activity on actin assembly at the leading edge. In GFP knockin animals, SRC-1 and MIG-13 distribute along the entire plasma membrane of migrating cells. We reveal that a receptor-like tyrosine phosphatase, PTP-3, maintains the F-actin polarity during neuroblast migration. Recombinant PTP-3 dephosphorylates SRC-1-dependent MIG-13 phosphorylation in vitro. Importantly, the endogenous PTP-3 accumulates at the rear of the migrating neuroblast, and its extracellular domain is essential for directional cell migration. We provide evidence that the asymmetrically localized tyrosine phosphatase PTP-3 spatially restricts MIG-13/Lrp12 receptor activity in migrating cells.

Keywords: cell polarity; cytoskeleton; directional cell migration; tyrosine kinase and phosphatase.

Conflict of interest statement
The authors declare no competing interest.

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47
Proc Natl Acad Sci U S A
. 2020 Jun 8;201916851. doi: 10.1073/pnas.1916851117. Online ahead of print.
Elimination of rNMPs From Mitochondrial DNA Has No Effect on Its Stability
Paulina H Wanrooij 1, Phong Tran 2, Liam J Thompson 3, Gustavo Carvalho 2, Sushma Sharma 2, Katrin Kreisel 3, Clara Navarrete 3, Anna-Lena Feldberg 2, Danielle L Watt 2, Anna Karin Nilsson 2, Martin K M Engqvist 3 4, Anders R Clausen 3, Andrei Chabes 1 5
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PMID: 32513727 DOI: 10.1073/pnas.1916851117
Free article
Abstract
Ribonucleotides (rNMPs) incorporated in the nuclear genome are a well-established threat to genome stability and can result in DNA strand breaks when not removed in a timely manner. However, the presence of a certain level of rNMPs is tolerated in mitochondrial DNA (mtDNA) although aberrant mtDNA rNMP content has been identified in disease models. We investigated the effect of incorporated rNMPs on mtDNA stability over the mouse life span and found that the mtDNA rNMP content increased during early life. The rNMP content of mtDNA varied greatly across different tissues and was defined by the rNTP/dNTP ratio of the tissue. Accordingly, mtDNA rNMPs were nearly absent in SAMHD1 -/- mice that have increased dNTP pools. The near absence of rNMPs did not, however, appreciably affect mtDNA copy number or the levels of mtDNA molecules with deletions or strand breaks in aged animals near the end of their life span. The physiological rNMP load therefore does not contribute to the progressive loss of mtDNA quality that occurs as mice age.

Keywords: SAMHD1; dNTP pool; mitochondrial DNA; mtDNA; ribonucleotide incorporation.

Copyright © 2020 the Author(s). Published by PNAS.

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The authors declare no competing interest.

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48
Proc Natl Acad Sci U S A
. 2020 Jun 8;201920197. doi: 10.1073/pnas.1920197117. Online ahead of print.
Evidence Generation, Decision Making, and Consequent Growth in Health Disparities
Anirban Basu 1 2, Kritee Gujral 3
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PMID: 32513684 DOI: 10.1073/pnas.1920197117
Abstract
Evidence is valuable because it informs decisions to produce better outcomes. However, the same evidence that is complete for some individuals or groups may be incomplete for others, leading to inefficiencies in decision making and growth in disparities in outcomes. Specifically, the presence of treatment effect heterogeneity across some measure of baseline risk, and noisy information about such heterogeneity, can induce self-selection into randomized clinical trials (RCTs) by patients with distributions of baseline risk different from that of the target population. Consequently, average results from RCTs can disproportionately affect the treatment choices of patients with different baseline risks. Using economic models for these sequential processes of RCT enrollment, information generation, and the resulting treatment choice decisions, we show that the dynamic consequences of such information flow and behaviors may lead to growth in disparities in health outcomes across racial and ethnic categories. These disparities arise due to either the differential distribution of risk across those categories at the time RCT results are reported or the different rate of change of baseline risk over time across race and ethnicity, even though the distribution of risk within the RCT matched that of the target population when the RCT was conducted. We provide evidence on how these phenomena may have contributed to the growth in racial disparity in diabetes incidence.

Keywords: diabetes incidence; evidence-based medicine; health disparity; treatment effect heterogeneity.

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The authors declare no competing interest.

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49
Proc Natl Acad Sci U S A
. 2020 Jun 8;201918696. doi: 10.1073/pnas.1918696117. Online ahead of print.
Predicting Optical Spectra for Optoelectronic Polymers Using Coarse-Grained Models and Recurrent Neural Networks
Lena Simine 1, Thomas C Allen 1, Peter J Rossky 2
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PMID: 32513725 DOI: 10.1073/pnas.1918696117
Abstract
Coarse-grained modeling of conjugated polymers has become an increasingly popular route to investigate the physics of organic optoelectronic materials. While ultraviolet (UV)-vis spectroscopy remains one of the key experimental methods for the interrogation of these materials, a rigorous bridge between simulated coarse-grained structures and spectroscopy has not been established. Here, we address this challenge by developing a method that can predict spectra of conjugated polymers directly from coarse-grained representations while avoiding repetitive procedures such as ad hoc back-mapping from coarse-grained to atomistic representations followed by spectral computation using quantum chemistry. Our approach is based on a generative deep-learning model: the long-short-term memory recurrent neural network (LSTM-RNN). The latter is suggested by the apparent similarity between natural languages and the mathematical structure of perturbative expansions of, in our case, excited-state energies perturbed by conformational fluctuations. We also use this model to explore the level of sensitivity of spectra to the coarse-grained representation back-mapping protocol. Our approach presents a tool uniquely suited for improving postsimulation analysis protocols, as well as, potentially, for including spectral data as input in the refinement of coarse-grained potentials.

Keywords: coarse-grained modeling; conjugated polymers; machine learning; molecular spectroscopy.

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The authors declare no competing interest.

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50
Proc Natl Acad Sci U S A
. 2020 Jun 8;201920725. doi: 10.1073/pnas.1920725117. Online ahead of print.
H1 Linker Histones Silence Repetitive Elements by Promoting Both Histone H3K9 Methylation and Chromatin Compaction
Sean E Healton 1, Hugo D Pinto 2, Laxmi N Mishra 2, Gregory A Hamilton 2 3, Justin C Wheat 2, Kalina Swist-Rosowska 4, Nicholas Shukeir 4, Yali Dou 5, Ulrich Steidl 2, Thomas Jenuwein 4, Matthew J Gamble 2 3, Arthur I Skoultchi 1
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PMID: 32513732 DOI: 10.1073/pnas.1920725117
Abstract
Nearly 50% of mouse and human genomes are composed of repetitive sequences. Transcription of these sequences is tightly controlled during development to prevent genomic instability, inappropriate gene activation and other maladaptive processes. Here, we demonstrate an integral role for H1 linker histones in silencing repetitive elements in mouse embryonic stem cells. Strong H1 depletion causes a profound de-repression of several classes of repetitive sequences, including major satellite, LINE-1, and ERV. Activation of repetitive sequence transcription is accompanied by decreased H3K9 trimethylation of repetitive sequence chromatin. H1 linker histones interact directly with Suv39h1, Suv39h2, and SETDB1, the histone methyltransferases responsible for H3K9 trimethylation of chromatin within these regions, and stimulate their activity toward chromatin in vitro. However, we also implicate chromatin compaction mediated by H1 as an additional, dominant repressive mechanism for silencing of repetitive major satellite sequences. Our findings elucidate two distinct, H1-mediated pathways for silencing heterochromatin.

Keywords: chromatin; epigenetics; linker histones; repetitive elements.

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The authors declare no competing interest.

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51
Proc Natl Acad Sci U S A
. 2020 Jun 8;202003898. doi: 10.1073/pnas.2003898117. Online ahead of print.
Oxidation Promoted Osmotic Energy Conversion in Black Phosphorus Membranes
Zhen Zhang 1 2, Panpan Zhang 1 2, Sheng Yang 1 2, Tao Zhang 1 2, Markus Löffler 3, Huanhuan Shi 1 2, Martin R Lohe 1 2, Xinliang Feng 4 2
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PMID: 32513735 DOI: 10.1073/pnas.2003898117
Abstract
Two-dimensional (2D) nanofluidic ion transporting membranes show great promise in harvesting the "blue" osmotic energy between river water and sea water. Black phosphorus (BP), an emerging layered material, has recently been explored for a wide range of ambient applications. However, little attention has been paid to the extraction of the worldwide osmotic energy, despite its large potential as an energy conversion membrane. Here, we report an experimental investigation of BP membrane in osmotic energy conversion and reveal how the oxidation of BP influences power generation. Through controllable oxidation in water, power output of the BP membrane can be largely enhanced, which can be attributed to the generated charged phosphorus compounds. Depending on the valence of oxidized BP that is associated with oxygen concentration, the power density can be precisely controlled and substantially promoted by ∼220% to 1.6 W/m2 (compared with the pristine BP membrane). Moreover, through constructing a heterostructure with graphene oxide, ion selectivity of the BP membrane increases by ∼80%, contributing to enhanced charge separation efficiency and thus improved performance of ∼4.7 W/m2 that outperforms most of the state-of-the-art 2D nanofluidic membranes.

Keywords: black phosphorus; ion transport; nanofluidics; osmotic energy.

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The authors declare no competing interest.

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52
Proc Natl Acad Sci U S A
. 2020 Jun 8;202001324. doi: 10.1073/pnas.2001324117. Online ahead of print.
FtsK in Motion Reveals Its Mechanism for Double-Stranded DNA Translocation
Nicolas L Jean 1, Trevor J Rutherford 1, Jan Löwe 2
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PMID: 32513722 DOI: 10.1073/pnas.2001324117
Free article
Abstract
FtsK protein contains a fast DNA motor that is involved in bacterial chromosome dimer resolution. During cell division, FtsK translocates double-stranded DNA until both dif recombination sites are placed at mid cell for subsequent dimer resolution. Here, we solved the 3.6-Å resolution electron cryo-microscopy structure of the motor domain of FtsK while translocating on its DNA substrate. Each subunit of the homo-hexameric ring adopts a unique conformation and one of three nucleotide states. Two DNA-binding loops within four subunits form a pair of spiral staircases within the ring, interacting with the two DNA strands. This suggests that simultaneous conformational changes in all ATPase domains at each catalytic step generate movement through a mechanism related to filament treadmilling. While the ring is only rotating around the DNA slowly, it is instead the conformational states that rotate around the ring as the DNA substrate is pushed through.

Keywords: DNA translocation; bacterial cell division; chromosome segregation; cryo-EM.

Copyright © 2020 the Author(s). Published by PNAS.

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The authors declare no competing interest.

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53
Proc Natl Acad Sci U S A
. 2020 Jun 8;202004664. doi: 10.1073/pnas.2004664117. Online ahead of print.
Sir2 Mitigates an Intrinsic Imbalance in Origin Licensing Efficiency Between Early- And Late-Replicating Euchromatin
Timothy Hoggard 1, Carolin A Müller 2, Conrad A Nieduszynski 2, Michael Weinreich 3, Catherine A Fox 4
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PMID: 32513739 DOI: 10.1073/pnas.2004664117
Abstract
A eukaryotic chromosome relies on the function of multiple spatially distributed DNA replication origins for its stable inheritance. The spatial location of an origin is determined by the chromosomal position of an MCM complex, the inactive form of the DNA replicative helicase that is assembled onto DNA in G1-phase (also known as origin licensing). While the biochemistry of origin licensing is understood, the mechanisms that promote an adequate spatial distribution of MCM complexes across chromosomes are not. We have elucidated a role for the Sir2 histone deacetylase in establishing the normal distribution of MCM complexes across Saccharomyces cerevisiae chromosomes. In the absence of Sir2, MCM complexes accumulated within both early-replicating euchromatin and telomeric heterochromatin, and replication activity within these regions was enhanced. Concomitantly, the duplication of several regions of late-replicating euchromatin were delayed. Thus, Sir2-mediated attenuation of origin licensing within both euchromatin and telomeric heterochromatin established the normal spatial distribution of origins across yeast chromosomes important for normal genome duplication.

Keywords: Sir; chromatin; chromosomes; origin licensing; yeast.

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The authors declare no competing interest.

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54
Proc Natl Acad Sci U S A
. 2020 Jun 8;202001384. doi: 10.1073/pnas.2001384117. Online ahead of print.
Establishing Rod Shape From Spherical, Peptidoglycan-Deficient Bacterial Spores
Huan Zhang 1, Garrett A Mulholland 1, Sofiene Seef 2, Shiwei Zhu 3 4, Jun Liu 3 4, Tâm Mignot 2, Beiyan Nan 5
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PMID: 32513721 DOI: 10.1073/pnas.2001384117
Abstract
Chemical-induced spores of the Gram-negative bacterium Myxococcus xanthus are peptidoglycan (PG)-deficient. It is unclear how these spherical spores germinate into rod-shaped, walled cells without preexisting PG templates. We found that germinating spores first synthesize PG randomly on spherical surfaces. MglB, a GTPase-activating protein, forms a cluster that responds to the status of PG growth and stabilizes at one future cell pole. Following MglB, the Ras family GTPase MglA localizes to the second pole. MglA directs molecular motors to transport the bacterial actin homolog MreB and the Rod PG synthesis complexes away from poles. The Rod system establishes rod shape de novo by elongating PG at nonpolar regions. Thus, similar to eukaryotic cells, the interactions between GTPase, cytoskeletons, and molecular motors initiate spontaneous polarization in bacteria.

Keywords: GTPase; MreB; Rod system; germination; gliding motor.

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The authors declare no competing interest.

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55
Proc Natl Acad Sci U S A
. 2020 Jun 8;201916206. doi: 10.1073/pnas.1916206117. Online ahead of print.
Reversible Suppression of T Cell Function in the Bone Marrow Microenvironment of Acute Myeloid Leukemia
Adam J Lamble 1, Yoko Kosaka 2, Ted Laderas 3, Allie Maffit 1, Andy Kaempf 4, Lauren K Brady 5, Weiwei Wang 6, Nicola Long 7, Jennifer N Saultz 8, Motomi Mori 4, David Soong 5, Clare V LeFave 9, Fei Huang 5, Homer Adams 3rd 5, Marc M Loriaux 7 8, Cristina E Tognon 7, Pierrette Lo 7, Jeffrey W Tyner 7 10, Guang Fan 11, Shannon K McWeeney 3 7, Brian J Druker 12 8 13, Evan F Lind 14 7 10
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PMID: 32513686 DOI: 10.1073/pnas.1916206117
Free article
Abstract
Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with approximately four new cases per 100,000 persons per year. Standard treatment for AML consists of induction chemotherapy with remission achieved in 50 to 75% of cases. Unfortunately, most patients will relapse and die from their disease, as 5-y survival is roughly 29%. Therefore, other treatment options are urgently needed. In recent years, immune-based therapies have led to unprecedented rates of survival among patients with some advanced cancers. Suppression of T cell function in the tumor microenvironment is commonly observed and may play a role in AML. We found that there is a significant association between T cell infiltration in the bone marrow microenvironment of newly diagnosed patients with AML and increased overall survival. Functional studies aimed at establishing the degree of T cell suppression in patients with AML revealed impaired T cell function in many patients. In most cases, T cell proliferation could be restored by blocking the immune checkpoint molecules PD-1, CTLA-4, or TIM3. Our data demonstrate that AML establishes an immune suppressive environment in the bone marrow, in part through T cell checkpoint function.

Keywords: AML; T cell; checkpoint blockade; immune microenvironment; leukemia.

Copyright © 2020 the Author(s). Published by PNAS.

Conflict of interest statement
Competing interest statement: L.K.B., F.H., D.S., and H.A. are employees of Janssen Pharmaceuticals R&D, LLC. D.S. is currently an employee and holds stock in Genmab. C.V.L is an employee of Lab Connect LLC. J.N.S. receives research support form Kyn Therapeutics. B.J.D. has the following disclosures: Scientific Advisory Board for Aileron Therapeutics, ALLCRON, Cepheid, Vivid Biosciences, Celgene, RUNX1 Research Program, EnLiven Therapeutics, Gilead Sciences (inactive), Baxalta (inactive), Monojul (inactive); Scientific Advisory Board and Stock: Aptose Biosciences, Blueprint Medicines, Beta Cat, Iterion Therapeutics, Third Coast Therapeutics, GRAIL (inactive), CTI BioPharma (inactive); Scientific Founder: MolecularMD (inactive, acquired by ICON); Board of Directors and Stock: Amgen; Board of Directors: Burroughs Wellcome Fund, CureOne; Joint Steering Committee: Beat AML LLS; Founder: VB Therapeutics; Clinical Trial Funding: Novartis, Bristol-Myers Squibb, Pfizer; royalties from Patent 6958335 (Novartis exclusive license) and Oregon Health & Science University and Dana-Farber Cancer Institute (one Merck exclusive license). J.W.T. receives research support from Aptose, Array, AstraZeneca, Constellation, Genentech, Gilead, Incyte, Janssen, Petra, Seattle Genetics, Syros, and Takeda. J.W.T. is a cofounder of Leap Oncology. E.F.L. receives research support from Janssen Pharmaceuticals, Celgene Amgen, and Kyn Therapeutics.

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56
Proc Natl Acad Sci U S A
. 2020 Jun 8;202008601. doi: 10.1073/pnas.2008601117. Online ahead of print.
Are the Hippocampus and Its Network Necessary for Creativity?
Roberto Cabeza 1 2, Maxi Becker 2, Simon W Davis 3 4
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PMID: 32513728 DOI: 10.1073/pnas.2008601117
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The authors declare no competing interest.

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57
Proc Natl Acad Sci U S A
. 2020 Jun 8;202000508. doi: 10.1073/pnas.2000508117. Online ahead of print.
Peripheral Myelin Protein 22 Preferentially Partitions Into Ordered Phase Membrane Domains
Justin T Marinko 1 2, Anne K Kenworthy 3 4, Charles R Sanders 5 2 6
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PMID: 32513719 DOI: 10.1073/pnas.2000508117
Free article
Abstract
The ordered environment of cholesterol-rich membrane nanodomains is thought to exclude many transmembrane (TM) proteins. Nevertheless, some multispan helical transmembrane proteins have been proposed to partition into these environments. Here, giant plasma membrane vesicles (GPMVs) were employed to quantitatively show that the helical tetraspan peripheral myelin protein 22 (PMP22) exhibits a pronounced preference for, promotes the formation of, and stabilizes ordered membrane domains. Neither S-palmitoylation of PMP22 nor its putative cholesterol binding motifs are required for this preference. In contrast, Charcot-Marie-Tooth disease-causing mutations that disrupt the stability of PMP22 tertiary structure reduce or eliminate this preference in favor of the disordered phase. These studies demonstrate that the ordered phase preference of PMP22 derives from global structural features associated with the folded form of this protein, providing a glimpse at the structural factors that promote raft partitioning for multispan helical membrane proteins.

Keywords: membrane phase domain; ordered; peripheral myelin protein 22.

Copyright © 2020 the Author(s). Published by PNAS.

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The authors declare no competing interest.

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58
Proc Natl Acad Sci U S A
. 2020 Jun 8;202000265. doi: 10.1073/pnas.2000265117. Online ahead of print.
Direct Imaging of Rapid Tethering of Synaptic Vesicles Accompanying Exocytosis at a Fast Central Synapse
Takafumi Miki 1 2, Mitsuharu Midorikawa 3, Takeshi Sakaba 1
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PMID: 32513685 DOI: 10.1073/pnas.2000265117
Abstract
A high rate of synaptic vesicle (SV) release is required at cerebellar mossy fiber terminals for rapid information processing. As the number of release sites is limited, fast SV reloading is necessary to achieve sustained release. However, rapid reloading has not been observed directly. Here, we visualize SV movements near presynaptic membrane using total internal reflection fluorescence (TIRF) microscopy. Upon stimulation, SVs appeared in the TIRF-field and became tethered to the presynaptic membrane with unexpectedly rapid time course, almost as fast as SVs disappeared due to release. However, such stimulus-induced tethering was abolished by inhibiting exocytosis, suggesting that the tethering is tightly coupled to preceding exocytosis. The newly tethered vesicles became fusion competent not immediately but only 300 ms to 400 ms after tethering. Together with model simulations, we propose that rapid tethering leads to an immediate filling of vacated spaces and release sites within <100 nm of the active zone by SVs, which serve as precursors of readily releasable vesicles, thereby shortening delays during sustained activity.

Keywords: presynapse; synapse; synaptic vesicle; vesicle recruitment.

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The authors declare no competing interest.

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59
Proc Natl Acad Sci U S A
. 2020 Jun 8;201921301. doi: 10.1073/pnas.1921301117. Online ahead of print.
Radiocarbon-based Approach Capable of Subannual Precision Resolves the Origins of the Site of Por-Bajin
Margot Kuitems 1, Andrei Panin 2 3, Andrea Scifo 4, Irina Arzhantseva 5 6, Yury Kononov 3, Petra Doeve 7, Andreas Neocleous 8, Michael Dee 4
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PMID: 32513700 DOI: 10.1073/pnas.1921301117
Free article
Abstract
Inadequate resolution is the principal limitation of radiocarbon dating. However, recent work has shown that exact-year precision is attainable if use can be made of past increases in atmospheric radiocarbon concentration or so-called Miyake events. Here, this nascent method is applied to an archaeological site of previously unknown age. We locate the distinctive radiocarbon signal of the year 775 common era (CE) in wood from the base of the Uyghur monument of Por-Bajin in Russia. Our analysis shows that the construction of Por-Bajin started in the summer of 777 CE, a foundation date that resolves decades of debate and allows the origin and purpose of the building to be established.

Keywords: Miyake event; Por-Bajin; archaeology; exact-year precision; radiocarbon dating.

Copyright © 2020 the Author(s). Published by PNAS.

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60
Proc Natl Acad Sci U S A
. 2020 Jun 8;201919887. doi: 10.1073/pnas.1919887117. Online ahead of print.
Hypothalamic Tanycytes Generate Acute Hyperphagia Through Activation of the Arcuate Neuronal Network
Matei Bolborea 1, Eric Pollatzek 2, Heather Benford 2, Tamara Sotelo-Hitschfeld 2, Nicholas Dale 1
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PMID: 32513737 DOI: 10.1073/pnas.1919887117
Free article
Abstract
Hypothalamic tanycytes are chemosensitive glial cells that contact the cerebrospinal fluid in the third ventricle and send processes into the hypothalamic parenchyma. To test whether they can activate neurons of the arcuate nucleus, we targeted expression of a Ca2+-permeable channelrhodopsin (CatCh) specifically to tanycytes. Activation of tanycytes ex vivo depolarized orexigenic (neuropeptide Y/agouti-related protein; NPY/AgRP) and anorexigenic (proopiomelanocortin; POMC) neurons via an ATP-dependent mechanism. In vivo, activation of tanycytes triggered acute hyperphagia only in the fed state during the inactive phase of the light-dark cycle.

Keywords: ATP; arcuate nucleus; food intake; hypothalamus; tanycyte.

Copyright © 2020 the Author(s). Published by PNAS.

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The authors declare no competing interest.

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61
Published Erratum Proc Natl Acad Sci U S A
. 2020 Jun 8;202009782. doi: 10.1073/pnas.2009782117. Online ahead of print.
Correction for Tomaskovic-Devey Et Al., Rising Between-Workplace Inequalities in High-Income Countries
No authors listed
PMID: 32513705 DOI: 10.1073/pnas.2009782117
Erratum for
Rising between-workplace inequalities in high-income countries.
Tomaskovic-Devey D, Rainey A, Avent-Holt D, Bandelj N, Boza I, Cort D, Godechot O, Hajdu G, Hällsten M, Henriksen LF, Hermansen AS, Hou F, Jung J, Kanjuo-Mrčela A, King J, Kodama N, Kristal T, Křížková A, Lippényi Z, Melzer SM, Mun E, Penner A, Petersen T, Poje A, Safi M, Thaning M, Tufail Z.
Proc Natl Acad Sci U S A. 2020 Apr 28;117(17):9277-9283. doi: 10.1073/pnas.1918249117. Epub 2020 Apr 13.
PMID: 32284412 Free PMC article.
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62
Published Erratum Proc Natl Acad Sci U S A
. 2020 Jun 8;202009131. doi: 10.1073/pnas.2009131117. Online ahead of print.
Correction for Alexander-Bloch Et Al., Imaging Local Genetic Influences on Cortical Folding
No authors listed
PMID: 32513695 DOI: 10.1073/pnas.2009131117
Erratum for
Imaging local genetic influences on cortical folding.
Alexander-Bloch AF, Raznahan A, Vandekar SN, Seidlitz J, Lu Z, Matthias SR, Knowles E, Mollon J, Rodrigue A, Curran JE, Görring HHH, Satterthwaite TD, Gur RE, Bassett DS, Hoftman GD, Pearlson G, Shinohara RT, Liu S, Fox PT, Almasy L, Blangero J, Glahn DC.
Proc Natl Acad Sci U S A. 2020 Mar 31;117(13):7430-7436. doi: 10.1073/pnas.1912064117. Epub 2020 Mar 13.
PMID: 32170019 Free PMC article.
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63
Published Erratum Proc Natl Acad Sci U S A
. 2020 Jun 8;202010013. doi: 10.1073/pnas.2010013117. Online ahead of print.
Correction for Ram Et Al., Predicting Microbial Growth in a Mixed Culture From Growth Curve Data
No authors listed
PMID: 32513691 DOI: 10.1073/pnas.2010013117
Erratum for
Predicting microbial growth in a mixed culture from growth curve data.
Ram Y, Dellus-Gur E, Bibi M, Karkare K, Obolski U, Feldman MW, Cooper TF, Berman J, Hadany L.
Proc Natl Acad Sci U S A. 2019 Jul 16;116(29):14698-14707. doi: 10.1073/pnas.1902217116. Epub 2019 Jun 28.
PMID: 31253703 Free PMC article.
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64
Published Erratum Proc Natl Acad Sci U S A
. 2020 Jun 8;202009107. doi: 10.1073/pnas.2009107117. Online ahead of print.
Correction for Jiao Et Al., Statin-induced GGPP Depletion Blocks Macropinocytosis and Starves Cells With Oncogenic Defects
No authors listed
PMID: 32513707 DOI: 10.1073/pnas.2009107117
Erratum for
Statin-induced GGPP depletion blocks macropinocytosis and starves cells with oncogenic defects.
Jiao Z, Cai H, Long Y, Sirka OK, Padmanaban V, Ewald AJ, Devreotes PN.
Proc Natl Acad Sci U S A. 2020 Feb 25;117(8):4158-4168. doi: 10.1073/pnas.1917938117. Epub 2020 Feb 12.
PMID: 32051246
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65
Published Erratum Proc Natl Acad Sci U S A
. 2020 Jun 8;202008788. doi: 10.1073/pnas.2008788117. Online ahead of print.
Correction for Sear at Al., Human Settlement of East Polynesia Earlier, Incremental, and Coincident With Prolonged South Pacific Drought
No authors listed
PMID: 32513740 DOI: 10.1073/pnas.2008788117
Erratum for
Human settlement of East Polynesia earlier, incremental, and coincident with prolonged South Pacific drought.
Sear DA, Allen MS, Hassall JD, Maloney AE, Langdon PG, Morrison AE, Henderson ACG, Mackay H, Croudace IW, Clarke C, Sachs JP, Macdonald G, Chiverrell RC, Leng MJ, Cisneros-Dozal LM, Fonville T.
Proc Natl Acad Sci U S A. 2020 Apr 21;117(16):8813-8819. doi: 10.1073/pnas.1920975117. Epub 2020 Apr 6.
PMID: 32253300 Free PMC article.
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