PLOS Biology (new articles)

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Structure of the DP1–DP2 PolD complex bound with DNA and its implications for the evolutionary history of DNA and RNA polymerases

сб, 2019-01-19 00:00

by Pierre Raia, Marta Carroni, Etienne Henry, Gérard Pehau-Arnaudet, Sébastien Brûlé, Pierre Béguin, Ghislaine Henneke, Erik Lindahl, Marc Delarue, Ludovic Sauguet

PolD is an archaeal replicative DNA polymerase (DNAP) made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). Recently, we reported the individual crystal structures of the DP1 and DP2 catalytic cores, thereby revealing that PolD is an atypical DNAP that has all functional properties of a replicative DNAP but with the catalytic core of an RNA polymerase (RNAP). We now report the DNA-bound cryo–electron microscopy (cryo-EM) structure of the heterodimeric DP1–DP2 PolD complex from Pyrococcus abyssi, revealing a unique DNA-binding site. Comparison of PolD and RNAPs extends their structural similarities and brings to light the minimal catalytic core shared by all cellular transcriptases. Finally, elucidating the structure of the PolD DP1–DP2 interface, which is conserved in all eukaryotic replicative DNAPs, clarifies their evolutionary relationships with PolD and sheds light on the domain acquisition and exchange mechanism that occurred during the evolution of the eukaryotic replisome.
Категории: Biology, Journals

Avoidance response to CO<sub>2</sub> in the lateral horn

пт, 2019-01-18 00:00

by Nélia Varela, Miguel Gaspar, Sophie Dias, Maria Luísa Vasconcelos

In flies, the olfactory information is carried from the first relay in the brain, the antennal lobe, to the mushroom body (MB) and the lateral horn (LH). Olfactory associations are formed in the MB. The LH was ascribed a role in innate responses based on the stereotyped connectivity with the antennal lobe, stereotyped physiological responses to odors, and MB silencing experiments. Direct evidence for the functional role of the LH is still missing. Here, we investigate the behavioral role of the LH neurons (LHNs) directly, using the CO2 response as a paradigm. Our results show the involvement of the LH in innate responses. Specifically, we demonstrate that activity in two sets of neurons is required for the full behavioral response to CO2. Tests of the behavioral response to other odors indicate the neurons are selective to CO2 response. Using calcium imaging, we observe that the two sets of neurons respond to CO2 in a different manner. Using independent manipulation and recording of the two sets of neurons, we find that the one that projects to the superior intermediate protocerebrum (SIP) also outputs to the local neurons within the LH. The design of simultaneous output at the LH and the SIP, an output of the MB, allows for coordination between innate and learned responses.
Категории: Biology, Journals

Organic cation transporter 3 (Oct3) is a distinct catecholamines clearance route in adipocytes mediating the beiging of white adipose tissue

пт, 2019-01-18 00:00

by Wenxin Song, Qi Luo, Yuping Zhang, Linkang Zhou, Ye Liu, Zhilong Ma, Jianan Guo, Yuedong Huang, Lili Cheng, Ziyi Meng, Zicheng Li, Bin Zhang, Siqi Li, Sook Wah Yee, Hao Fan, Peng Li, Kathleen M. Giacomini, Ligong Chen

Beiging of white adipose tissue (WAT) is a particularly appealing target for therapeutics in the treatment of metabolic diseases through norepinephrine (NE)-mediated signaling pathways. Although previous studies report NE clearance mechanisms via SLC6A2 on sympathetic neurons or proinflammatory macrophages in adipose tissues (ATs), the low catecholamine clearance capacity of SLC6A2 may limit the cleaning efficiency. Here, we report that mouse organic cation transporter 3 (Oct3; Slc22a3) is highly expressed in WAT and displays the greatest uptake rate of NE as a selective non-neural route of NE clearance in white adipocytes, which differs from other known routes such as adjacent neurons or macrophages. We further show that adipocytes express high levels of NE degradation enzymes Maoa, Maob, and Comt, providing the molecular basis on NE clearance by adipocytes together with its reuptake transporter Oct3. Under NE administration, ablation of Oct3 induces higher body temperature, thermogenesis, and lipolysis compared with littermate controls. After prolonged cold challenge, inguinal WAT (ingWAT) in adipose-specific Oct3-deficient mice shows much stronger browning characteristics and significantly elevated expression of thermogenic and mitochondrial biogenesis genes than in littermate controls, and this response involves enhanced β-adrenergic receptor (β-AR)/protein kinase A (PKA)/cyclic adenosine monophosphate (cAMP)-responsive element binding protein (Creb) pathway activation. Glycolytic genes are reprogrammed to significantly higher levels to compensate for the loss of ATP production in adipose-specific Oct3 knockout (KO) mice, indicating the fundamental role of glucose metabolism during beiging. Inhibition of β-AR largely abolishes the higher lipolytic and thermogenic activities in Oct3-deficient ingWAT, indicating the NE overload in the vicinity of adipocytes in Oct3 KO adipocytes. Of note, reduced functional alleles in human OCT3 are also identified to be associated with increased basal metabolic rate (BMR). Collectively, our results demonstrate that Oct3 governs β-AR activity as a NE recycling transporter in white adipocytes, offering potential therapeutic applications for metabolic disorders.
Категории: Biology, Journals

CD4 occupancy triggers sequential pre-fusion conformational states of the HIV-1 envelope trimer with relevance for broadly neutralizing antibody activity

чт, 2019-01-17 00:00

by Branislav Ivan, Zhaozhi Sun, Harini Subbaraman, Nikolas Friedrich, Alexandra Trkola

During the entry process, the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer undergoes a sequence of conformational changes triggered by both CD4 and coreceptor engagement. Resolving the conformation of these transient entry intermediates has proven challenging. Here, we fine-mapped the antigenicity of entry intermediates induced by increasing CD4 engagement of cell surface–expressed Env. Escalating CD4 triggering led to the sequential adoption of different pre-fusion conformational states of the Env trimer, up to the pre-hairpin conformation, that we assessed for antibody epitope presentation. Maximal accessibility of the coreceptor binding site was detected below Env saturation by CD4. Exposure of the fusion peptide and heptad repeat 1 (HR1) required higher CD4 occupancy. Analyzing the diverse antigenic states of the Env trimer, we obtained key insights into the transitions in epitope accessibility of broadly neutralizing antibodies (bnAbs). Several bnAbs preferentially bound CD4-triggered Env, indicating a potential capacity to neutralize both pre- and post-CD4 engagement, which needs to be explored. Assessing binding and neutralization activity of bnAbs, we confirm antibody dissociation rates as a driver of incomplete neutralization. Collectively, our findings highlight a need to resolve Env conformations that are neutralization-relevant to provide guidance for immunogen development.
Категории: Biology, Journals

Large carnivores under assault in Alaska

ср, 2019-01-16 00:00

by William J. Ripple, Sterling D. Miller, John W. Schoen, Sanford P. Rabinowitch

In Alaska, gray wolves (Canis lupis), brown bears (Ursus arctos), and black bears (U. americanus) are managed in most of the state in ways intended to significantly reduce their abundance in the expectation of increasing hunter harvests of ungulates. To our knowledge, Alaska is unique in the world because this management priority is both widespread and mandated by state law. Large carnivore management in Alaska is a reversion to outdated management concepts and occurs without effective monitoring programs designed to scientifically evaluate impacts on predator populations. Large carnivore management in Alaska should be based on rigorous science including the status and trends of carnivore populations.
Категории: Biology, Journals

Adding function to the genome of African <i>Salmonella</i> Typhimurium ST313 strain D23580

ср, 2019-01-16 00:00

by Rocío Canals, Disa L. Hammarlöf, Carsten Kröger, Siân V. Owen, Wai Yee Fong, Lizeth Lacharme-Lora, Xiaojun Zhu, Nicolas Wenner, Sarah E. Carden, Jared Honeycutt, Denise M. Monack, Robert A. Kingsley, Philip Brownridge, Roy R. Chaudhuri, Will P. M. Rowe, Alexander V. Predeus, Karsten Hokamp, Melita A. Gordon, Jay C. D. Hinton

Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.
Категории: Biology, Journals

The brown algal mode of tip growth: Keeping stress under control

вт, 2019-01-15 00:00

by Hervé Rabillé, Bernard Billoud, Benoit Tesson, Sophie Le Panse, Élodie Rolland, Bénédicte Charrier

Tip growth has been studied in pollen tubes, root hairs, and fungal and oomycete hyphae and is the most widely distributed unidirectional growth process on the planet. It ensures spatial colonization, nutrient predation, fertilization, and symbiosis with growth speeds of up to 800 μm h−1. Although turgor-driven growth is intuitively conceivable, a closer examination of the physical processes at work in tip growth raises a paradox: growth occurs where biophysical forces are low, because of the increase in curvature in the tip. All tip-growing cells studied so far rely on the modulation of cell wall extensibility via the polarized excretion of cell wall–loosening compounds at the tip. Here, we used a series of quantitative measurements at the cellular level and a biophysical simulation approach to show that the brown alga Ectocarpus has an original tip-growth mechanism. In this alga, the establishment of a steep gradient in cell wall thickness can compensate for the variation in tip curvature, thereby modulating wall stress within the tip cell. Bootstrap analyses support the robustness of the process, and experiments with fluorescence recovery after photobleaching (FRAP) confirmed the active vesicle trafficking in the shanks of the apical cell, as inferred from the model. In response to auxin, biophysical measurements change in agreement with the model. Although we cannot strictly exclude the involvement of a gradient in mechanical properties in Ectocarpus morphogenesis, the viscoplastic model of cell wall mechanics strongly suggests that brown algae have evolved an alternative strategy of tip growth. This strategy is largely based on the control of cell wall thickness rather than fluctuations in cell wall mechanical properties.
Категории: Biology, Journals

Resolving the apparent transmission paradox of African sleeping sickness

сб, 2019-01-12 00:00

by Paul Capewell, Katie Atkins, William Weir, Vincent Jamonneau, Mamadou Camara, Caroline Clucas, Nono-Raymond K. Swar, Dieudonne M. Ngoyi, Brice Rotureau, Paul Garside, Alison P. Galvani, Bruno Bucheton, Annette MacLeod

Human African trypanosomiasis (HAT), or African sleeping sickness, is a fatal disease found throughout sub-Saharan Africa. The disease is close to elimination in many areas, although it was similarly close to elimination once before and subsequently reemerged, despite seemingly low rates of transmission. Determining how these foci persisted and overcame an apparent transmission paradox is key to finally eliminating HAT. By assessing clinical, laboratory, and mathematical data, we propose that asymptomatic infections contribute to transmission through the presence of an overlooked reservoir of skin-dwelling parasites. Our assessment suggests that a combination of asymptomatic and parasitaemic cases is sufficient to maintain transmission at foci without animal reservoirs, and we argue that the current policy not to treat asymptomatic HAT should be reconsidered.
Категории: Biology, Journals

Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation

пт, 2019-01-11 00:00

by Phil Oberacker, Peter Stepper, Donna M. Bond, Sven Höhn, Jule Focken, Vivien Meyer, Luca Schelle, Victoria J. Sugrue, Gert-Jan Jeunen, Tim Moser, Steven R. Hore, Ferdinand von Meyenn, Katharina Hipp, Timothy A. Hore, Tomasz P. Jurkowski

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.
Категории: Biology, Journals

<i>Drosophila melanogaster</i> cloak their eggs with pheromones, which prevents cannibalism

пт, 2019-01-11 00:00

by Sunitha Narasimha, Konstantin O. Nagornov, Laure Menin, Antonio Mucciolo, Astrid Rohwedder, Bruno M. Humbel, Martin Stevens, Andreas S. Thum, Yury O. Tsybin, Roshan K. Vijendravarma

Oviparous animals across many taxa have evolved diverse strategies that deter egg predation, providing valuable tests of how natural selection mitigates direct fitness loss. Communal egg laying in nonsocial species minimizes egg predation. However, in cannibalistic species, this very behavior facilitates egg predation by conspecifics (cannibalism). Similarly, toxins and aposematic signaling that deter egg predators are often inefficient against resistant conspecifics. Egg cannibalism can be adaptive, wherein cannibals may benefit through reduced competition and added nutrition, but since it reduces Darwinian fitness, the evolution of anticannibalistic strategies is rife. However, such strategies are likely to be nontoxic because deploying toxins against related individuals would reduce inclusive fitness. Here, we report how D. melanogaster use specific hydrocarbons to chemically mask their eggs from cannibal larvae. Using an integrative approach combining behavioral, sensory, and mass spectrometry methods, we demonstrate that maternally provisioned pheromone 7,11-heptacosadiene (7,11-HD) in the eggshell’s wax layer deters egg cannibalism. Furthermore, we show that 7,11-HD is nontoxic, can mask underlying substrates (for example, yeast) when coated upon them, and its detection requires pickpocket 23 (ppk23) gene function. Finally, using light and electron microscopy, we demonstrate how maternal pheromones leak-proof the egg, consequently concealing it from conspecific larvae. Our data suggest that semiochemicals possibly subserve in deceptive functions across taxa, especially when predators rely on chemical cues to forage, and stimulate further research on deceptive strategies mediated through nonvisual sensory modules. This study thus highlights how integrative approaches can illuminate our understanding on the adaptive significance of deceptive defenses and the mechanisms through which they operate.
Категории: Biology, Journals

Annotated primary scientific literature: A pedagogical tool for undergraduate courses

чт, 2019-01-10 00:00

by Matthew Kararo, Melissa McCartney

Annotated primary scientific literature is a teaching and learning resource that provides scaffolding for undergraduate students acculturating to the authentic scientific practice of obtaining and evaluating information through the medium of primary scientific literature. Utilizing annotated primary scientific literature as an integrated pedagogical tool could enable more widespread use of primary scientific literature in undergraduate science classrooms with minimal disruption to existing syllabi. Research is ongoing to determine an optimal implementation protocol, with these preliminary iterations presented here serving as a first look at how students respond to annotated primary scientific literature. The undergraduate biology student participants in our study did not, in general, have an abundance of experience reading primary scientific literature; however, they found the annotations useful, especially for vocabulary and graph interpretation. We present here an implementation protocol for using annotated primary literature in the classroom that minimizes the use of valuable classroom time and requires no additional pedagogical training for instructors.
Категории: Biology, Journals

Identification and characterization of a mosquito-specific eggshell organizing factor in <i>Aedes aegypti</i> mosquitoes

ср, 2019-01-09 00:00

by Jun Isoe, Lauren E. Koch, Yurika E. Isoe, Alberto A. Rascón Jr., Heidi E. Brown, Brooke B. Massani, Roger L. Miesfeld

Mosquito-borne diseases are responsible for several million human deaths annually around the world. One approach to controlling mosquito populations is to disrupt molecular processes or antagonize novel metabolic targets required for the production of viable eggs. To this end, we focused our efforts on identifying proteins required for completion of embryonic development that are mosquito selective and represent potential targets for vector control. We performed bioinformatic analyses to identify putative protein-coding sequences that are specific to mosquito genomes. Systematic RNA interference (RNAi) screening of 40 mosquito-specific genes was performed by injecting double-stranded RNA (dsRNA) into female Aedes aegypti mosquitoes. This experimental approach led to the identification of eggshell organizing factor 1 (EOF1, AAEL012336), which plays an essential role in the formation and melanization of the eggshell. Eggs deposited by EOF1-deficient mosquitoes have nonmelanized fragile eggshells, and all embryos are nonviable. Scanning electron microscopy (SEM) analysis identified that exochorionic eggshell structures are strongly affected in EOF1-deficient mosquitoes. EOF1 is a potential novel target, to our knowledge, for exploring the identification and development of mosquito-selective and biosafe small-molecule inhibitors.
Категории: Biology, Journals

A transient helix in the disordered region of dynein light intermediate chain links the motor to structurally diverse adaptors for cargo transport

вт, 2019-01-08 00:00

by Ricardo Celestino, Morkos A. Henen, José B. Gama, Cátia Carvalho, Maxwell McCabe, Daniel J. Barbosa, Alexandra Born, Parker J. Nichols, Ana X. Carvalho, Reto Gassmann, Beat Vögeli

All animal cells use the motor cytoplasmic dynein 1 (dynein) to transport diverse cargo toward microtubule minus ends and to organize and position microtubule arrays such as the mitotic spindle. Cargo-specific adaptors engage with dynein to recruit and activate the motor, but the molecular mechanisms remain incompletely understood. Here, we use structural and dynamic nuclear magnetic resonance (NMR) analysis to demonstrate that the C-terminal region of human dynein light intermediate chain 1 (LIC1) is intrinsically disordered and contains two short conserved segments with helical propensity. NMR titration experiments reveal that the first helical segment (helix 1) constitutes the main interaction site for the adaptors Spindly (SPDL1), bicaudal D homolog 2 (BICD2), and Hook homolog 3 (HOOK3). In vitro binding assays show that helix 1, but not helix 2, is essential in both LIC1 and LIC2 for binding to SPDL1, BICD2, HOOK3, RAB-interacting lysosomal protein (RILP), RAB11 family-interacting protein 3 (RAB11FIP3), ninein (NIN), and trafficking kinesin-binding protein 1 (TRAK1). Helix 1 is sufficient to bind RILP, whereas other adaptors require additional segments preceding helix 1 for efficient binding. Point mutations in the C-terminal helix 1 of Caenorhabditis elegans LIC, introduced by genome editing, severely affect development, locomotion, and life span of the animal and disrupt the distribution and transport kinetics of membrane cargo in axons of mechanosensory neurons, identical to what is observed when the entire LIC C-terminal region is deleted. Deletion of the C-terminal helix 2 delays dynein-dependent spindle positioning in the one-cell embryo but overall does not significantly perturb dynein function. We conclude that helix 1 in the intrinsically disordered region of LIC provides a conserved link between dynein and structurally diverse cargo adaptor families that is critical for dynein function in vivo.
Категории: Biology, Journals

Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in <i>Trichomonas vaginalis</i>

сб, 2019-01-05 00:00

by Abhijith Makki, Petr Rada, Vojtěch Žárský, Sami Kereïche, Lubomír Kováčik, Marian Novotný, Tobias Jores, Doron Rapaport, Jan Tachezy

Mitochondria originated from proteobacterial endosymbionts, and their transition to organelles was tightly linked to establishment of the protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, an unexpected diversity of subunits between eukaryotic lineages has been predicted. However, experimental knowledge is limited to a few organisms, and so far, it remains unsettled whether the triplet-pore or the twin-pore structure is the generic form of TOM complex. Here, we analysed the TOM complex in hydrogenosomes, a metabolically specialised anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. We demonstrate that the highly divergent β-barrel T. vaginalis TOM (TvTom)40-2 forms a translocation channel to conduct hydrogenosomal protein import. TvTom40-2 is present in high molecular weight complexes, and their analysis revealed the presence of four tail-anchored (TA) proteins. Two of them, Tom36 and Tom46, with heat shock protein (Hsp)20 and tetratricopeptide repeat (TPR) domains, can bind hydrogenosomal preproteins and most likely function as receptors. A third subunit, Tom22-like protein, has a short cis domain and a conserved Tom22 transmembrane segment but lacks a trans domain. The fourth protein, hydrogenosomal outer membrane protein 19 (Homp19) has no known homology. Furthermore, our data indicate that TvTOM is associated with sorting and assembly machinery (Sam)50 that is involved in β-barrel assembly. Visualisation of TvTOM by electron microscopy revealed that it forms three pores and has an unconventional skull-like shape. Although TvTOM seems to lack Tom7, our phylogenetic profiling predicted Tom7 in free-living excavates. Collectively, our results suggest that the triplet-pore TOM complex, composed of three conserved subunits, was present in the last common eukaryotic ancestor (LECA), while receptors responsible for substrate binding evolved independently in different eukaryotic lineages.
Категории: Biology, Journals

Differential and convergent utilization of autophagy components by positive-strand RNA viruses

сб, 2019-01-05 00:00

by Emma Abernathy, Roberto Mateo, Karim Majzoub, Nick van Buuren, Sara W. Bird, Jan E. Carette, Karla Kirkegaard

Many viruses interface with the autophagy pathway, a highly conserved process for recycling cellular components. For three viral infections in which autophagy constituents are proviral (poliovirus, dengue, and Zika), we developed a panel of knockouts (KOs) of autophagy-related genes to test which components of the canonical pathway are utilized. We discovered that each virus uses a distinct set of initiation components; however, all three viruses utilize autophagy-related gene 9 (ATG9), a lipid scavenging protein, and LC3 (light-chain 3), which is involved in membrane curvature. These results show that viruses use noncanonical routes for membrane sculpting and LC3 recruitment. By measuring viral RNA abundance, we also found that poliovirus utilizes these autophagy components for intracellular growth, while dengue and Zika virus only use autophagy components for post-RNA replication processes. Comparing how RNA viruses manipulate the autophagy pathway reveals new noncanonical autophagy routes, explains the exacerbation of disease by starvation, and uncovers common targets for antiviral drugs.
Категории: Biology, Journals

Contest models highlight inherent inefficiencies of scientific funding competitions

чт, 2019-01-03 00:00

by Kevin Gross, Carl T. Bergstrom

Scientific research funding is allocated largely through a system of soliciting and ranking competitive grant proposals. In these competitions, the proposals themselves are not the deliverables that the funder seeks, but instead are used by the funder to screen for the most promising research ideas. Consequently, some of the funding program's impact on science is squandered because applying researchers must spend time writing proposals instead of doing science. To what extent does the community's aggregate investment in proposal preparation negate the scientific impact of the funding program? Are there alternative mechanisms for awarding funds that advance science more efficiently? We use the economic theory of contests to analyze how efficiently grant proposal competitions advance science, and compare them with recently proposed, partially randomized alternatives such as lotteries. We find that the effort researchers waste in writing proposals may be comparable to the total scientific value of the research that the funding supports, especially when only a few proposals can be funded. Moreover, when professional pressures motivate investigators to seek funding for reasons that extend beyond the value of the proposed science (e.g., promotion, prestige), the entire program can actually hamper scientific progress when the number of awards is small. We suggest that lost efficiency may be restored either by partial lotteries for funding or by funding researchers based on past scientific success instead of proposals for future work.
Категории: Biology, Journals

Enabling precision medicine via standard communication of HTS provenance, analysis, and results

вт, 2019-01-01 00:00

by Gil Alterovitz, Dennis Dean, Carole Goble, Michael R. Crusoe, Stian Soiland-Reyes, Amanda Bell, Anais Hayes, Anita Suresh, Anjan Purkayastha, Charles H. King, Dan Taylor, Elaine Johanson, Elaine E. Thompson, Eric Donaldson, Hiroki Morizono, Hsinyi Tsang, Jeet K. Vora, Jeremy Goecks, Jianchao Yao, Jonas S. Almeida, Jonathon Keeney, KanakaDurga Addepalli, Konstantinos Krampis, Krista M. Smith, Lydia Guo, Mark Walderhaug, Marco Schito, Matthew Ezewudo, Nuria Guimera, Paul Walsh, Robel Kahsay, Srikanth Gottipati, Timothy C. Rodwell, Toby Bloom, Yuching Lai, Vahan Simonyan, Raja Mazumder

A personalized approach based on a patient's or pathogen’s unique genomic sequence is the foundation of precision medicine. Genomic findings must be robust and reproducible, and experimental data capture should adhere to findable, accessible, interoperable, and reusable (FAIR) guiding principles. Moreover, effective precision medicine requires standardized reporting that extends beyond wet-lab procedures to computational methods. The BioCompute framework (https://w3id.org/biocompute/1.3.0) enables standardized reporting of genomic sequence data provenance, including provenance domain, usability domain, execution domain, verification kit, and error domain. This framework facilitates communication and promotes interoperability. Bioinformatics computation instances that employ the BioCompute framework are easily relayed, repeated if needed, and compared by scientists, regulators, test developers, and clinicians. Easing the burden of performing the aforementioned tasks greatly extends the range of practical application. Large clinical trials, precision medicine, and regulatory submissions require a set of agreed upon standards that ensures efficient communication and documentation of genomic analyses. The BioCompute paradigm and the resulting BioCompute Objects (BCOs) offer that standard and are freely accessible as a GitHub organization (https://github.com/biocompute-objects) following the “Open-Stand.org principles for collaborative open standards development.” With high-throughput sequencing (HTS) studies communicated using a BCO, regulatory agencies (e.g., Food and Drug Administration [FDA]), diagnostic test developers, researchers, and clinicians can expand collaboration to drive innovation in precision medicine, potentially decreasing the time and cost associated with next-generation sequencing workflow exchange, reporting, and regulatory reviews.
Категории: Biology, Journals

Catalytic mechanism of the tyrosinase reaction toward the Tyr<sup>98</sup> residue in the caddie protein

вт, 2019-01-01 00:00

by Yasuyuki Matoba, Shogo Kihara, Naohiko Bando, Hironari Yoshitsu, Miyuki Sakaguchi, Kure’e Kayama, Sachiko Yanagisawa, Takashi Ogura, Masanori Sugiyama

Tyrosinase (EC 1.14.18.1), a copper-containing monooxygenase, catalyzes the conversion of phenol to the corresponding ortho-quinone. The Streptomyces tyrosinase is generated as a complex with a “caddie” protein that facilitates the transport of two copper ions into the active center. In our previous study, the Tyr98 residue in the caddie protein, which is accommodated in the pocket of active center of tyrosinase, has been found to be converted to a reactive quinone through the formations of the μ-η2:η2-peroxo-dicopper(II) and Cu(II)-dopasemiquinone intermediates. Until now—despite extensive studies for the tyrosinase reaction based on the crystallographic analysis, low-molecular-weight models, and computer simulations—the catalytic mechanism has been unable to be made clear at an atomic level. To make the catalytic mechanism of tyrosinase clear, in the present study, the cryo-trapped crystal structures were determined at very high resolutions (1.16–1.70 Å). The structures suggest the existence of an important step for the tyrosinase reaction that has not yet been found: that is, the hydroxylation reaction is triggered by the movement of CuA, which induces the syn-to-anti rearrangement of the copper ligands after the formation of μ-η2:η2-peroxo-dicopper(II) core. By the rearrangement, the hydroxyl group of the substrate is placed in an equatorial position, allowing the electrophilic attack to the aromatic ring by the Cu2O2 oxidant.
Категории: Biology, Journals

Podoplanin<sup>+</sup> tumor lymphatics are rate limiting for breast cancer metastasis

сб, 2018-12-29 00:00

by Yang Chen, Doruk Keskin, Hikaru Sugimoto, Keizo Kanasaki, Patricia E. Phillips, Lauren Bizarro, Arlene Sharpe, Valerie S. LeBleu, Raghu Kalluri

Metastatic dissemination employs both the blood and lymphatic vascular systems. Solid tumors dynamically remodel and generate both vessel types during cancer progression. Lymphatic vessel invasion and cancer cells in the tumor-draining lymph nodes (LNs) are prognostic markers for breast cancer metastasis and patient outcome, and tumor-induced lymphangiogenesis likely influences metastasis. Deregulated tumor tissue fluid homeostasis and immune trafficking associated with tumor lymphangiogenesis may contribute to metastatic spreading; however, the precise functional characterization of lymphatic endothelial cells (LECs) in tumors is challenged by the lack of specific reagents to decipher their rate-limiting role in metastasis. Therefore, we generated novel transgenic mice (PDPN promoter-driven Cre recombinase transgene [PDPN-Cre] and PDPN promoter-driven thymidine kinase transgene [PDPN-tk]) that allow for the identification and genetically controlled depletion of proliferating podoplanin (Pdpn)-expressing LECs. We demonstrate that suppression of lymphangiogenesis is successfully achieved in lymphangioma lesions induced in the PDPN-tk mice. In multiple metastatic breast cancer mouse models, we identified distinct roles for LECs in primary and metastatic tumors. Our findings support the functional contribution of primary tumor lymphangiogenesis in controlling metastasis to axillary LNs and lung parenchyma. Reduced lymphatic vessel density enhanced primary tumor lymphedema and increased the frequency of intratumoral macrophages but was not associated with a significant impact on primary tumor growth despite a marked reduction in metastatic dissemination. Our findings identify the rate-limiting contribution of the breast tumor lymphatic vessels for lung metastasis.
Категории: Biology, Journals

An opposing function of paralogs in balancing developmental synapse maturation

чт, 2018-12-27 00:00

by Plinio D. Favaro, Xiaojie Huang, Leon Hosang, Sophia Stodieck, Lei Cui, Yu-zhang Liu, Karl-Alexander Engelhardt, Frank Schmitz, Yan Dong, Siegrid Löwel, Oliver M. Schlüter

The disc-large (DLG)–membrane-associated guanylate kinase (MAGUK) family of proteins forms a central signaling hub of the glutamate receptor complex. Among this family, some proteins regulate developmental maturation of glutamatergic synapses, a process vulnerable to aberrations, which may lead to neurodevelopmental disorders. As is typical for paralogs, the DLG-MAGUK proteins postsynaptic density (PSD)-95 and PSD-93 share similar functional domains and were previously thought to regulate glutamatergic synapses similarly. Here, we show that they play opposing roles in glutamatergic synapse maturation. Specifically, PSD-95 promoted, whereas PSD-93 inhibited maturation of immature α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid–type glutamate receptor (AMPAR)–silent synapses in mouse cortex during development. Furthermore, through experience-dependent regulation of its protein levels, PSD-93 directly inhibited PSD-95’s promoting effect on silent synapse maturation in the visual cortex. The concerted function of these two paralogs governed the critical period of juvenile ocular dominance plasticity (jODP), and fine-tuned visual perception during development. In contrast to the silent synapse–based mechanism of adjusting visual perception, visual acuity improved by different mechanisms. Thus, by controlling the pace of silent synapse maturation, the opposing but properly balanced actions of PSD-93 and PSD-95 are essential for fine-tuning cortical networks for receptive field integration during developmental critical periods, and imply aberrations in either direction of this process as potential causes for neurodevelopmental disorders.
Категории: Biology, Journals