Pub Date : 2021-04-23DOI: 10.1101/2021.04.22.441049
N. Kono, Hiroyuki Nakamura, Masaru Mori, Yuki Yoshida, Rintaro Ohtoshi, A. Malay, Daniel A Pedrazzoli Moran, M. Tomita, K. Numata, K. Arakawa
Significance Artificial synthesis of spider silk has been actively pursued. However, until now, the natural mechanical properties of spider silk have been largely unreproducible. We thoroughly investigated the genomes and transcripts of four related species of orb-weaver spiders as well as the proteins in their silk threads. Then, in addition to spidroin, we found several low-molecular-weight proteins in common. Interestingly, the low-molecular-weight protein component of spider dragline silk doubled the tensile strength of artificial silk–based material. This discovery will greatly advance the industry and research on the use of protein-based materials. Dragline silk of golden orb-weaver spiders (Nephilinae) is noted for its unsurpassed toughness, combining extraordinary extensibility and tensile strength, suggesting industrial application as a sustainable biopolymer material. To pinpoint the molecular composition of dragline silk and the roles of its constituents in achieving its mechanical properties, we report a multiomics approach, combining high-quality genome sequencing and assembly, silk gland transcriptomics, and dragline silk proteomics of four Nephilinae spiders. We observed the consistent presence of the MaSp3B spidroin unique to this subfamily as well as several nonspidroin SpiCE proteins. Artificial synthesis and the combination of these components in vitro showed that the multicomponent nature of dragline silk, including MaSp3B and SpiCE, along with MaSp1 and MaSp2, is essential to realize the mechanical properties of spider dragline silk.
{"title":"Multicomponent nature underlies the extraordinary mechanical properties of spider dragline silk","authors":"N. Kono, Hiroyuki Nakamura, Masaru Mori, Yuki Yoshida, Rintaro Ohtoshi, A. Malay, Daniel A Pedrazzoli Moran, M. Tomita, K. Numata, K. Arakawa","doi":"10.1101/2021.04.22.441049","DOIUrl":"https://doi.org/10.1101/2021.04.22.441049","url":null,"abstract":"Significance Artificial synthesis of spider silk has been actively pursued. However, until now, the natural mechanical properties of spider silk have been largely unreproducible. We thoroughly investigated the genomes and transcripts of four related species of orb-weaver spiders as well as the proteins in their silk threads. Then, in addition to spidroin, we found several low-molecular-weight proteins in common. Interestingly, the low-molecular-weight protein component of spider dragline silk doubled the tensile strength of artificial silk–based material. This discovery will greatly advance the industry and research on the use of protein-based materials. Dragline silk of golden orb-weaver spiders (Nephilinae) is noted for its unsurpassed toughness, combining extraordinary extensibility and tensile strength, suggesting industrial application as a sustainable biopolymer material. To pinpoint the molecular composition of dragline silk and the roles of its constituents in achieving its mechanical properties, we report a multiomics approach, combining high-quality genome sequencing and assembly, silk gland transcriptomics, and dragline silk proteomics of four Nephilinae spiders. We observed the consistent presence of the MaSp3B spidroin unique to this subfamily as well as several nonspidroin SpiCE proteins. Artificial synthesis and the combination of these components in vitro showed that the multicomponent nature of dragline silk, including MaSp3B and SpiCE, along with MaSp1 and MaSp2, is essential to realize the mechanical properties of spider dragline silk.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"71 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76398159","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2021-04-22DOI: 10.1101/2021.04.21.440847
T. Jay, Yunsik Kang, A. Jefferson, M. Freeman
Significance Forward genetic screens in Drosophila have played an integral role in elucidating cellular and molecular pathways that govern almost every facet of biology. However, current screening methods in Drosophila are either fast, but limited in their specificity, or rely on imaging, requiring substantial expertise, time, and cost. We developed a rapid GFP-based ELISA that, when paired with the wealth of genetic tools available in Drosophila, can be used to screen for regulators of many subpopulations of cells, transcriptional programs, and proteins. Using this assay, we identified genes required for astrocytic synapse elimination. This technique provides a screening platform that is fast, accessible, and broadly applicable to many pathways and processes, making Drosophila an even more powerful screening platform. Drosophila is a powerful model in which to perform genetic screens, but screening assays that are both rapid and can be used to examine a wide variety of cellular and molecular pathways are limited. Drosophila offer an extensive toolbox of GFP-based transcriptional reporters, GFP-tagged proteins, and driver lines, which can be used to express GFP in numerous subpopulations of cells. Thus, a tool that can rapidly and quantitatively evaluate GFP levels in Drosophila tissue would provide a broadly applicable screening platform. We developed a GFP-based enzyme-linked immunosorbent assay (ELISA) that can detect GFP in Drosophila lysates collected from whole animals and dissected tissues across all stages of Drosophila development. We demonstrate that this assay can detect membrane-localized GFP in a variety of neuronal and glial populations and validate that it can identify genes that change the morphology of these cells, as well as changes in STAT and JNK transcriptional activity. We found that this assay can detect endogenously GFP-tagged proteins, including Draper, Cryptochrome, and the synaptic marker Brp. This approach is able to detect changes in Brp-GFP signal during developmental synaptic remodeling, and known genetic regulators of glial synaptic engulfment could be identified using this ELISA method. Finally, we used the assay to perform a small-scale screen, which identified Syntaxins as potential regulators of astrocyte-mediated synapse elimination. Together, these studies establish an ELISA as a rapid, easy, and quantitative in vivo screening method that can be used to assay a wide breadth of fundamental biological questions.
{"title":"An ELISA-based method for rapid genetic screens in Drosophila","authors":"T. Jay, Yunsik Kang, A. Jefferson, M. Freeman","doi":"10.1101/2021.04.21.440847","DOIUrl":"https://doi.org/10.1101/2021.04.21.440847","url":null,"abstract":"Significance Forward genetic screens in Drosophila have played an integral role in elucidating cellular and molecular pathways that govern almost every facet of biology. However, current screening methods in Drosophila are either fast, but limited in their specificity, or rely on imaging, requiring substantial expertise, time, and cost. We developed a rapid GFP-based ELISA that, when paired with the wealth of genetic tools available in Drosophila, can be used to screen for regulators of many subpopulations of cells, transcriptional programs, and proteins. Using this assay, we identified genes required for astrocytic synapse elimination. This technique provides a screening platform that is fast, accessible, and broadly applicable to many pathways and processes, making Drosophila an even more powerful screening platform. Drosophila is a powerful model in which to perform genetic screens, but screening assays that are both rapid and can be used to examine a wide variety of cellular and molecular pathways are limited. Drosophila offer an extensive toolbox of GFP-based transcriptional reporters, GFP-tagged proteins, and driver lines, which can be used to express GFP in numerous subpopulations of cells. Thus, a tool that can rapidly and quantitatively evaluate GFP levels in Drosophila tissue would provide a broadly applicable screening platform. We developed a GFP-based enzyme-linked immunosorbent assay (ELISA) that can detect GFP in Drosophila lysates collected from whole animals and dissected tissues across all stages of Drosophila development. We demonstrate that this assay can detect membrane-localized GFP in a variety of neuronal and glial populations and validate that it can identify genes that change the morphology of these cells, as well as changes in STAT and JNK transcriptional activity. We found that this assay can detect endogenously GFP-tagged proteins, including Draper, Cryptochrome, and the synaptic marker Brp. This approach is able to detect changes in Brp-GFP signal during developmental synaptic remodeling, and known genetic regulators of glial synaptic engulfment could be identified using this ELISA method. Finally, we used the assay to perform a small-scale screen, which identified Syntaxins as potential regulators of astrocyte-mediated synapse elimination. Together, these studies establish an ELISA as a rapid, easy, and quantitative in vivo screening method that can be used to assay a wide breadth of fundamental biological questions.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78731830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2021-04-20DOI: 10.1101/2021.04.20.440636
A. S. Kamenik, I. Singh, P. Lak, T. E. Balius, K. Liedl, B. Shoichet
Significance The dynamic nature of biomolecules is typically neglected in docking screens for ligand discovery. The key to benefitting from various receptor conformations is not only structural but also thermodynamic information. Here, we test a general approach that uses conformational preferences from enhanced and conventional molecular dynamics simulations to account for the cost of transitions to high-energy states. Including this information as a conformational penalty term in a docking, scoring function, we perform retrospective and prospective screens and experimentally confirm predicted ligands with Tm upshift and X-ray crystallography. This not only allows us to test the predicted ligands for binding, it also tests whether they bind to the conformation of the binding site for which they were predicted. Protein flexibility remains a major challenge in library docking because of difficulties in sampling conformational ensembles with accurate probabilities. Here, we use the model cavity site of T4 lysozyme L99A to test flexible receptor docking with energy penalties from molecular dynamics (MD) simulations. Crystallography with larger and smaller ligands indicates that this cavity can adopt three major conformations: open, intermediate, and closed. Since smaller ligands typically bind better to the cavity site, we anticipate an energy penalty for the cavity opening. To estimate its magnitude, we calculate conformational preferences from MD simulations. We find that including a penalty term is essential for retrospective ligand enrichment; otherwise, high-energy states dominate the docking. We then prospectively docked a library of over 900,000 compounds for new molecules binding to each conformational state. Absent a penalty term, the open conformation dominated the docking results; inclusion of this term led to a balanced sampling of ligands against each state. High ranked molecules were experimentally tested by Tm upshift and X-ray crystallography. From 33 selected molecules, we identified 18 ligands and determined 13 crystal structures. Most interesting were those bound to the open cavity, where the buried site opens to bulk solvent. Here, highly unusual ligands for this cavity had been predicted, including large ligands with polar tails; these were confirmed both by binding and by crystallography. In docking, incorporating protein flexibility with thermodynamic weightings may thus access new ligand chemotypes. The MD approach to accessing and, crucially, weighting such alternative states may find general applicability.
{"title":"Energy penalties enhance flexible receptor docking in a model cavity","authors":"A. S. Kamenik, I. Singh, P. Lak, T. E. Balius, K. Liedl, B. Shoichet","doi":"10.1101/2021.04.20.440636","DOIUrl":"https://doi.org/10.1101/2021.04.20.440636","url":null,"abstract":"Significance The dynamic nature of biomolecules is typically neglected in docking screens for ligand discovery. The key to benefitting from various receptor conformations is not only structural but also thermodynamic information. Here, we test a general approach that uses conformational preferences from enhanced and conventional molecular dynamics simulations to account for the cost of transitions to high-energy states. Including this information as a conformational penalty term in a docking, scoring function, we perform retrospective and prospective screens and experimentally confirm predicted ligands with Tm upshift and X-ray crystallography. This not only allows us to test the predicted ligands for binding, it also tests whether they bind to the conformation of the binding site for which they were predicted. Protein flexibility remains a major challenge in library docking because of difficulties in sampling conformational ensembles with accurate probabilities. Here, we use the model cavity site of T4 lysozyme L99A to test flexible receptor docking with energy penalties from molecular dynamics (MD) simulations. Crystallography with larger and smaller ligands indicates that this cavity can adopt three major conformations: open, intermediate, and closed. Since smaller ligands typically bind better to the cavity site, we anticipate an energy penalty for the cavity opening. To estimate its magnitude, we calculate conformational preferences from MD simulations. We find that including a penalty term is essential for retrospective ligand enrichment; otherwise, high-energy states dominate the docking. We then prospectively docked a library of over 900,000 compounds for new molecules binding to each conformational state. Absent a penalty term, the open conformation dominated the docking results; inclusion of this term led to a balanced sampling of ligands against each state. High ranked molecules were experimentally tested by Tm upshift and X-ray crystallography. From 33 selected molecules, we identified 18 ligands and determined 13 crystal structures. Most interesting were those bound to the open cavity, where the buried site opens to bulk solvent. Here, highly unusual ligands for this cavity had been predicted, including large ligands with polar tails; these were confirmed both by binding and by crystallography. In docking, incorporating protein flexibility with thermodynamic weightings may thus access new ligand chemotypes. The MD approach to accessing and, crucially, weighting such alternative states may find general applicability.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"2675 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90839127","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2021-04-19DOI: 10.1101/2021.04.19.440349
M. Aramesh, Diana Stoycheva, I. Sandu, Stephan J. Ihle, Tamara Zünd, Jau-Ye Shiu, Csaba Forró, Mohammad Asghari, Margherita Bernero, Sebastian Lickert, A. Oxenius, V. Vogel, Enrico Klotzsch
Significance Microvilli are used by immune cells to sense the surface features of pathogens and antigen presenting cells. However, microvilli’s contribution in T cell signaling and activation is largely unknown. Here, we introduce a material-based platform for induction of microvilli formation in T cells, in which the dimensions of the microvilli can be controlled by tuning the dimensions of the nanotopographical features, such as pore depth, pore size, and interpore distance. We demonstrate the direct causality between microvilli formation and altered gene expression in T cells. We discover that the size of the microvilli critically influences T cell receptor agonistic independent signaling in T cells. The results provide a physical strategy for T cell activation and expansion for immunotherapy applications. T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanism by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200-nm pores but not in 400-nm pores. Consequently, formation of TCR nanoclustered hotspots within 200-nm pores allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.
{"title":"Nanoconfinement of microvilli alters gene expression and boosts T cell activation","authors":"M. Aramesh, Diana Stoycheva, I. Sandu, Stephan J. Ihle, Tamara Zünd, Jau-Ye Shiu, Csaba Forró, Mohammad Asghari, Margherita Bernero, Sebastian Lickert, A. Oxenius, V. Vogel, Enrico Klotzsch","doi":"10.1101/2021.04.19.440349","DOIUrl":"https://doi.org/10.1101/2021.04.19.440349","url":null,"abstract":"Significance Microvilli are used by immune cells to sense the surface features of pathogens and antigen presenting cells. However, microvilli’s contribution in T cell signaling and activation is largely unknown. Here, we introduce a material-based platform for induction of microvilli formation in T cells, in which the dimensions of the microvilli can be controlled by tuning the dimensions of the nanotopographical features, such as pore depth, pore size, and interpore distance. We demonstrate the direct causality between microvilli formation and altered gene expression in T cells. We discover that the size of the microvilli critically influences T cell receptor agonistic independent signaling in T cells. The results provide a physical strategy for T cell activation and expansion for immunotherapy applications. T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanism by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200-nm pores but not in 400-nm pores. Consequently, formation of TCR nanoclustered hotspots within 200-nm pores allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"56 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80417981","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2021-04-12DOI: 10.26434/CHEMRXIV.14394746.V1
A. Patra, Jerome Davis, Saran Pidaparthy, Manohar H. Karigerasi, B. Zahiri, A. Kulkarni, Michael A. Caple, D. Shoemaker, J. Zuo, P. Braun
Significance Layered sodium transition metal oxides constitute an important class of materials with applications including electrochemical energy storage, high-temperature superconductivity, and electrocatalysis. However, electrodeposition of these compounds, an approach commonly used to grow oxides, has been elusive due to their atmosphere instability and intrinsic incompatibility with aqueous electrolytes. Using a molten sodium hydroxide electrolyte, we demonstrate electrodeposition of O3 (O′3)- and P2-type layered sodium transition metal oxides and apply these electrodeposits as high areal capacity cathodes in sodium-ion batteries. The electrodeposits are micrometers thick, polycrystalline, and structurally similar to materials synthesized classically at high temperature. This work enables fabrication of previously inaccessible alkali and alkaline earth ion intercalated, higher valent transition group oxides in important thick film form factors. We introduce an intermediate-temperature (350 °C) dry molten sodium hydroxide-mediated binder-free electrodeposition process to grow the previously electrochemically inaccessible air- and moisture-sensitive layered sodium transition metal oxides, NaxMO2 (M = Co, Mn, Ni, Fe), in both thin and thick film form, compounds which are conventionally synthesized in powder form by solid-state reactions at temperatures ≥700 °C. As a key motivation for this work, several of these oxides are of interest as cathode materials for emerging sodium-ion–based electrochemical energy storage systems. Despite the low synthesis temperature and short reaction times, our electrodeposited oxides retain the key structural and electrochemical performance observed in high-temperature bulk synthesized materials. We demonstrate that tens of micrometers thick >75% dense NaxCoO2 and NaxMnO2 can be deposited in under 1 h. When used as cathodes for sodium-ion batteries, these materials exhibit near theoretical gravimetric capacities, chemical diffusion coefficients of Na+ ions (∼10−12 cm2⋅s−1), and high reversible areal capacities in the range ∼0.25 to 0.76 mA⋅h⋅cm−2, values significantly higher than those reported for binder-free sodium cathodes deposited by other techniques. The method described here resolves longstanding intrinsic challenges associated with traditional aqueous solution-based electrodeposition of ceramic oxides and opens a general solution chemistry approach for electrochemical processing of hitherto unexplored air- and moisture-sensitive high valent multinary structures with extended frameworks.
{"title":"Electrodeposition of atmosphere-sensitive ternary sodium transition metal oxide films for sodium-based electrochemical energy storage","authors":"A. Patra, Jerome Davis, Saran Pidaparthy, Manohar H. Karigerasi, B. Zahiri, A. Kulkarni, Michael A. Caple, D. Shoemaker, J. Zuo, P. Braun","doi":"10.26434/CHEMRXIV.14394746.V1","DOIUrl":"https://doi.org/10.26434/CHEMRXIV.14394746.V1","url":null,"abstract":"Significance Layered sodium transition metal oxides constitute an important class of materials with applications including electrochemical energy storage, high-temperature superconductivity, and electrocatalysis. However, electrodeposition of these compounds, an approach commonly used to grow oxides, has been elusive due to their atmosphere instability and intrinsic incompatibility with aqueous electrolytes. Using a molten sodium hydroxide electrolyte, we demonstrate electrodeposition of O3 (O′3)- and P2-type layered sodium transition metal oxides and apply these electrodeposits as high areal capacity cathodes in sodium-ion batteries. The electrodeposits are micrometers thick, polycrystalline, and structurally similar to materials synthesized classically at high temperature. This work enables fabrication of previously inaccessible alkali and alkaline earth ion intercalated, higher valent transition group oxides in important thick film form factors. We introduce an intermediate-temperature (350 °C) dry molten sodium hydroxide-mediated binder-free electrodeposition process to grow the previously electrochemically inaccessible air- and moisture-sensitive layered sodium transition metal oxides, NaxMO2 (M = Co, Mn, Ni, Fe), in both thin and thick film form, compounds which are conventionally synthesized in powder form by solid-state reactions at temperatures ≥700 °C. As a key motivation for this work, several of these oxides are of interest as cathode materials for emerging sodium-ion–based electrochemical energy storage systems. Despite the low synthesis temperature and short reaction times, our electrodeposited oxides retain the key structural and electrochemical performance observed in high-temperature bulk synthesized materials. We demonstrate that tens of micrometers thick >75% dense NaxCoO2 and NaxMnO2 can be deposited in under 1 h. When used as cathodes for sodium-ion batteries, these materials exhibit near theoretical gravimetric capacities, chemical diffusion coefficients of Na+ ions (∼10−12 cm2⋅s−1), and high reversible areal capacities in the range ∼0.25 to 0.76 mA⋅h⋅cm−2, values significantly higher than those reported for binder-free sodium cathodes deposited by other techniques. The method described here resolves longstanding intrinsic challenges associated with traditional aqueous solution-based electrodeposition of ceramic oxides and opens a general solution chemistry approach for electrochemical processing of hitherto unexplored air- and moisture-sensitive high valent multinary structures with extended frameworks.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"34 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81259838","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2021-04-09DOI: 10.1101/2021.04.06.21255017
Jietuo Wang, M. Alipour, Giovanni Soligo, Alessio Roccon, M. De Paoli, F. Picano, A. Soldati
Significance Violent expiratory events like coughs and sneezes represent an important route for the spread of respiratory viruses, such as SARS-CoV-2, the virus responsible for COVID-19. We use finely resolved experiments and simulations to quantify how the turbulent cloud of moist air exhaled during a sneeze largely increases the airborne time and the lifespan of virus-loaded droplets. By providing visualizations of the spatial distribution of the virus copies, we highlight the high infection risk associated with droplets that remain airborne in the near proximity of an infected individual. The present study aims at raising awareness among public health authorities about this infection risk, which is grossly underestimated by current guidelines. After the Spanish flu pandemic, it was apparent that airborne transmission was crucial to spreading virus contagion, and research responded by producing several fundamental works like the experiments of Duguid [J. P. Duguid, J. Hyg. 44, 6 (1946)] and the model of Wells [W. F. Wells, Am. J. Hyg. 20, 611–618 (1934)]. These seminal works have been pillars of past and current guidelines published by health organizations. However, in about one century, understanding of turbulent aerosol transport by jets and plumes has enormously progressed, and it is now time to use this body of developed knowledge. In this work, we use detailed experiments and accurate computationally intensive numerical simulations of droplet-laden turbulent puffs emitted during sneezes in a wide range of environmental conditions. We consider the same emission—number of drops, drop size distribution, and initial velocity—and we change environmental parameters such as temperature and humidity, and we observe strong variation in droplets’ evaporation or condensation in accordance with their local temperature and humidity microenvironment. We assume that 3% of the initial droplet volume is made of nonvolatile matter. Our systematic analysis confirms that droplets’ lifetime is always about one order of magnitude larger compared to previous predictions, in some cases up to 200 times. Finally, we have been able to produce original virus exposure maps, which can be a useful instrument for health scientists and practitioners to calibrate new guidelines to prevent short-range airborne disease transmission.
{"title":"Short-range exposure to airborne virus transmission and current guidelines","authors":"Jietuo Wang, M. Alipour, Giovanni Soligo, Alessio Roccon, M. De Paoli, F. Picano, A. Soldati","doi":"10.1101/2021.04.06.21255017","DOIUrl":"https://doi.org/10.1101/2021.04.06.21255017","url":null,"abstract":"Significance Violent expiratory events like coughs and sneezes represent an important route for the spread of respiratory viruses, such as SARS-CoV-2, the virus responsible for COVID-19. We use finely resolved experiments and simulations to quantify how the turbulent cloud of moist air exhaled during a sneeze largely increases the airborne time and the lifespan of virus-loaded droplets. By providing visualizations of the spatial distribution of the virus copies, we highlight the high infection risk associated with droplets that remain airborne in the near proximity of an infected individual. The present study aims at raising awareness among public health authorities about this infection risk, which is grossly underestimated by current guidelines. After the Spanish flu pandemic, it was apparent that airborne transmission was crucial to spreading virus contagion, and research responded by producing several fundamental works like the experiments of Duguid [J. P. Duguid, J. Hyg. 44, 6 (1946)] and the model of Wells [W. F. Wells, Am. J. Hyg. 20, 611–618 (1934)]. These seminal works have been pillars of past and current guidelines published by health organizations. However, in about one century, understanding of turbulent aerosol transport by jets and plumes has enormously progressed, and it is now time to use this body of developed knowledge. In this work, we use detailed experiments and accurate computationally intensive numerical simulations of droplet-laden turbulent puffs emitted during sneezes in a wide range of environmental conditions. We consider the same emission—number of drops, drop size distribution, and initial velocity—and we change environmental parameters such as temperature and humidity, and we observe strong variation in droplets’ evaporation or condensation in accordance with their local temperature and humidity microenvironment. We assume that 3% of the initial droplet volume is made of nonvolatile matter. Our systematic analysis confirms that droplets’ lifetime is always about one order of magnitude larger compared to previous predictions, in some cases up to 200 times. Finally, we have been able to produce original virus exposure maps, which can be a useful instrument for health scientists and practitioners to calibrate new guidelines to prevent short-range airborne disease transmission.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"28 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77094573","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Science and storytelling mean different things when they speak of truth. This difference leads some to blame storytelling for presenting a distorted view of science and contributing to misinformation. Yet others celebrate storytelling as a way to engage audiences and share accurate scientific information. This review disentangles the complexities of how storytelling intersects with scientific misinformation. Storytelling is the act of sharing a narrative, and science and narrative represent two distinct ways of constructing reality. Where science searches for broad patterns that capture general truths about the world, narratives search for connections through human experience that assign meaning and value to reality. I explore how these contrasting conceptions of truth manifest across different contexts to either promote or counter scientific misinformation. I also identify gaps in the literature and identify promising future areas of research. Even with their differences, the underlying purpose of both science and narrative seeks to make sense of the world and find our place within it. While narrative can indeed lead to scientific misinformation, narrative can also help science counter misinformation by providing meaning to reality that incorporates accurate science knowledge into human experience.
{"title":"The narrative truth about scientific misinformation","authors":"Michael F. Dahlstrom","doi":"10.2139/ssrn.3497784","DOIUrl":"https://doi.org/10.2139/ssrn.3497784","url":null,"abstract":"Science and storytelling mean different things when they speak of truth. This difference leads some to blame storytelling for presenting a distorted view of science and contributing to misinformation. Yet others celebrate storytelling as a way to engage audiences and share accurate scientific information. This review disentangles the complexities of how storytelling intersects with scientific misinformation. Storytelling is the act of sharing a narrative, and science and narrative represent two distinct ways of constructing reality. Where science searches for broad patterns that capture general truths about the world, narratives search for connections through human experience that assign meaning and value to reality. I explore how these contrasting conceptions of truth manifest across different contexts to either promote or counter scientific misinformation. I also identify gaps in the literature and identify promising future areas of research. Even with their differences, the underlying purpose of both science and narrative seeks to make sense of the world and find our place within it. While narrative can indeed lead to scientific misinformation, narrative can also help science counter misinformation by providing meaning to reality that incorporates accurate science knowledge into human experience.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"72 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72796375","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2021-04-06DOI: 10.1101/2021.04.06.438613
Kyle S. Skalenko, Lingting Li, Yuanchao Zhang, I. Vvedenskaya, Jared T. Winkelman, Alexander L. Cope, Deanne Taylor, Premal Shah, R. Ebright, J. Kinney, Yu Zhang, Bryce E. Nickels
Significance Primer-dependent transcription initiation—the use of RNA primers as initiating entities in transcription initiation—yields RNA products having a 5′-hydroxyl. Here, we show that primer-dependent initiation in vivo in Escherichia coli involves predominantly dinucleotide primers, involves any of the 16 possible dinucleotide primers, and depends on promoter sequences in, upstream, and downstream of the primer binding site. Crystal structures explain the structural basis of sequence dependence at the promoter position immediately upstream of the primer binding site, namely, interchain base stacking between the promoter template-strand nucleotide and primer 5′ nucleotide. Taken together, our findings provide a mechanistic and structural description of primer-dependent initiation in E. coli. Chemical modifications of RNA 5′-ends enable “epitranscriptomic” regulation, influencing multiple aspects of RNA fate. In transcription initiation, a large inventory of substrates compete with nucleoside triphosphates for use as initiating entities, providing an ab initio mechanism for altering the RNA 5′-end. In Escherichia coli cells, RNAs with a 5′-end hydroxyl are generated by use of dinucleotide RNAs as primers for transcription initiation, “primer-dependent initiation.” Here, we use massively systematic transcript end readout (MASTER) to detect and quantify RNA 5′-ends generated by primer-dependent initiation for ∼410 (∼1,000,000) promoter sequences in E. coli. The results show primer-dependent initiation in E. coli involves any of the 16 possible dinucleotide primers and depends on promoter sequences in, upstream, and downstream of the primer binding site. The results yield a consensus sequence for primer-dependent initiation, YTSS−2NTSS−1NTSSWTSS+1, where TSS is the transcription start site, NTSS−1NTSS is the primer binding site, Y is pyrimidine, and W is A or T. Biochemical and structure-determination studies show that the base pair (nontemplate-strand base:template-strand base) immediately upstream of the primer binding site (Y:RTSS−2, where R is purine) exerts its effect through the base on the DNA template strand (RTSS−2) through interchain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally encoded E. coli promoters support the conclusions from MASTER. Our findings provide a mechanistic and structural description of how TSS-region sequence hard-codes not only the TSS position but also the potential for epitranscriptomic regulation through primer-dependent transcription initiation.
{"title":"Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli","authors":"Kyle S. Skalenko, Lingting Li, Yuanchao Zhang, I. Vvedenskaya, Jared T. Winkelman, Alexander L. Cope, Deanne Taylor, Premal Shah, R. Ebright, J. Kinney, Yu Zhang, Bryce E. Nickels","doi":"10.1101/2021.04.06.438613","DOIUrl":"https://doi.org/10.1101/2021.04.06.438613","url":null,"abstract":"Significance Primer-dependent transcription initiation—the use of RNA primers as initiating entities in transcription initiation—yields RNA products having a 5′-hydroxyl. Here, we show that primer-dependent initiation in vivo in Escherichia coli involves predominantly dinucleotide primers, involves any of the 16 possible dinucleotide primers, and depends on promoter sequences in, upstream, and downstream of the primer binding site. Crystal structures explain the structural basis of sequence dependence at the promoter position immediately upstream of the primer binding site, namely, interchain base stacking between the promoter template-strand nucleotide and primer 5′ nucleotide. Taken together, our findings provide a mechanistic and structural description of primer-dependent initiation in E. coli. Chemical modifications of RNA 5′-ends enable “epitranscriptomic” regulation, influencing multiple aspects of RNA fate. In transcription initiation, a large inventory of substrates compete with nucleoside triphosphates for use as initiating entities, providing an ab initio mechanism for altering the RNA 5′-end. In Escherichia coli cells, RNAs with a 5′-end hydroxyl are generated by use of dinucleotide RNAs as primers for transcription initiation, “primer-dependent initiation.” Here, we use massively systematic transcript end readout (MASTER) to detect and quantify RNA 5′-ends generated by primer-dependent initiation for ∼410 (∼1,000,000) promoter sequences in E. coli. The results show primer-dependent initiation in E. coli involves any of the 16 possible dinucleotide primers and depends on promoter sequences in, upstream, and downstream of the primer binding site. The results yield a consensus sequence for primer-dependent initiation, YTSS−2NTSS−1NTSSWTSS+1, where TSS is the transcription start site, NTSS−1NTSS is the primer binding site, Y is pyrimidine, and W is A or T. Biochemical and structure-determination studies show that the base pair (nontemplate-strand base:template-strand base) immediately upstream of the primer binding site (Y:RTSS−2, where R is purine) exerts its effect through the base on the DNA template strand (RTSS−2) through interchain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally encoded E. coli promoters support the conclusions from MASTER. Our findings provide a mechanistic and structural description of how TSS-region sequence hard-codes not only the TSS position but also the potential for epitranscriptomic regulation through primer-dependent transcription initiation.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"40 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74652238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Alexander Sergeevich Spirin, an international member of the National Academy of Sciences, recited this poem by Boris Pasternak while lecturing to the bright minds nourished by him. Fatefully, he passed away on the snowy day of December 30, 2020, extinguishing one of the brightest candles, but leaving behind many others that he had set alight. Alex received his PhD degree from the A. N. Bach Institute of Biochemistry (Moscow) in 1957 under the mentorship of Andrey N. Belozersky, who in the 1930s discovered the universal occurrence of DNA in plants, previously assumed to exist only in animals. This was the time of the Khrushchev thaw and a period of intellectual exuberance in the Soviet Union and the world, with the discovery of the double helix by James Watson and Francis Crick. The first “student” whom Alex tutored in molecular biology was the President of the United Soviet Socialist Republic (USSR) Academy of Sciences, Mstislav Keldysh, who was greatly enchanted by the new and enlightening science. He granted Alex an opportunity to create a new Institute—the Institute of Protein Research—which was cofounded by Alex and Oleg Ptitsyn in 1967. It is located just outside Moscow in the town of Pushchino, a small academic center that had been especially built for biological research 10 years earlier. Alex’s philosophy was simple, but one often forsaken in modern science: Only scientific knowledge and the ability to envision new directions in research are meritorious, and the success of the individual depends on the teamwork of many. At his institution, one of the best known in the world dedicated to protein research, Alex created an intellectual hub, bringing together physicists, structural biologists, and biochemists. It was his strong belief that it is not past discovery, but the people and scientific culture that propel new directions in science. Alex and his team kept the fire of world-class science alive amid the blizzard of political and economic realities of the country that, until 1991, was the USSR. Alex was a member of the Russian Academy of Sciences, distinguished Professor and the head of Department of Molecular Biology of Moscow State University for almost half a century. His force of personality, his dedicated teaching, monographs, and textbooks—including the acclaimed Ribosomes (1)—shaped the directions of groundbreaking studies performed under his leadership or inspired by him. His lectures were renowned at the Faculty of Biology, Moscow State University, with students and researchers from around the city packing the auditorium to listen to Alex’s lectures on a myriad of topics, never repeating the same lecture twice. Alexander Spirin in the classroom. Image credit: Institute of Protein Research.
{"title":"Alexander Spirin (1931–2020): A visionary scientist, a teacher, a colleague, a friend","authors":"V. Evdokimova, Y. Svitkin, N. Sonenberg","doi":"10.1073/pnas.2103938118","DOIUrl":"https://doi.org/10.1073/pnas.2103938118","url":null,"abstract":"Alexander Sergeevich Spirin, an international member of the National Academy of Sciences, recited this poem by Boris Pasternak while lecturing to the bright minds nourished by him. Fatefully, he passed away on the snowy day of December 30, 2020, extinguishing one of the brightest candles, but leaving behind many others that he had set alight. Alex received his PhD degree from the A. N. Bach Institute of Biochemistry (Moscow) in 1957 under the mentorship of Andrey N. Belozersky, who in the 1930s discovered the universal occurrence of DNA in plants, previously assumed to exist only in animals. This was the time of the Khrushchev thaw and a period of intellectual exuberance in the Soviet Union and the world, with the discovery of the double helix by James Watson and Francis Crick. The first “student” whom Alex tutored in molecular biology was the President of the United Soviet Socialist Republic (USSR) Academy of Sciences, Mstislav Keldysh, who was greatly enchanted by the new and enlightening science. He granted Alex an opportunity to create a new Institute—the Institute of Protein Research—which was cofounded by Alex and Oleg Ptitsyn in 1967. It is located just outside Moscow in the town of Pushchino, a small academic center that had been especially built for biological research 10 years earlier. Alex’s philosophy was simple, but one often forsaken in modern science: Only scientific knowledge and the ability to envision new directions in research are meritorious, and the success of the individual depends on the teamwork of many. At his institution, one of the best known in the world dedicated to protein research, Alex created an intellectual hub, bringing together physicists, structural biologists, and biochemists. It was his strong belief that it is not past discovery, but the people and scientific culture that propel new directions in science. Alex and his team kept the fire of world-class science alive amid the blizzard of political and economic realities of the country that, until 1991, was the USSR. Alex was a member of the Russian Academy of Sciences, distinguished Professor and the head of Department of Molecular Biology of Moscow State University for almost half a century. His force of personality, his dedicated teaching, monographs, and textbooks—including the acclaimed Ribosomes (1)—shaped the directions of groundbreaking studies performed under his leadership or inspired by him. His lectures were renowned at the Faculty of Biology, Moscow State University, with students and researchers from around the city packing the auditorium to listen to Alex’s lectures on a myriad of topics, never repeating the same lecture twice. Alexander Spirin in the classroom. Image credit: Institute of Protein Research.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"20 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89639122","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2021-03-29DOI: 10.1017/S1431927621003299
H. Rahmani, Wen Ma, Zhongjun Hu, N. Daneshparvar, Dianne W. Taylor, J. McCammon, T. Irving, R. Edwards, K. Taylor
Significance Myosin II is the molecule that produces force in muscle contraction. Unlike the myosin head, its molecular motor, no atomic resolution structure of the ∼1000-residue–long α-helical coiled-coil tail has been reported. Here, we describe the cryo-EM atomic structure of the myosin tail within a native muscle thick filament. Three differences with crystal structures of myosin tail segments were found. The myosin head arrangement apparently alters the beginning of the tail. Striated muscle myosins have four skip residues, amino acids inserted to improve the alignment of charged residue clusters. Skips 1 and 3 agree with the crystal structures. Skip 2, which is a novel structure, and Skip 4 do not. Functional consequences are suggested by the myosin tail packing. The atomic structure of the complete myosin tail within thick filaments isolated from Lethocerus indicus flight muscle is described and compared to crystal structures of recombinant, human cardiac myosin tail segments. Overall, the agreement is good with three exceptions: the proximal S2, in which the filament has heads attached but the crystal structure doesn’t, and skip regions 2 and 4. At the head–tail junction, the tail α-helices are asymmetrically structured encompassing well-defined unfolding of 12 residues for one myosin tail, ∼4 residues of the other, and different degrees of α-helix unwinding for both tail α-helices, thereby providing an atomic resolution description of coiled-coil “uncoiling” at the head–tail junction. Asymmetry is observed in the nonhelical C termini; one C-terminal segment is intercalated between ribbons of myosin tails, the other apparently terminating at Skip 4 of another myosin tail. Between skip residues, crystal and filament structures agree well. Skips 1 and 3 also agree well and show the expected α-helix unwinding and coiled-coil untwisting in response to skip residue insertion. Skips 2 and 4 are different. Skip 2 is accommodated in an unusual manner through an increase in α-helix radius and corresponding reduction in rise/residue. Skip 4 remains helical in one chain, with the other chain unfolded, apparently influenced by the acidic myosin C terminus. The atomic model may shed some light on thick filament mechanosensing and is a step in understanding the complex roles that thick filaments of all species undergo during muscle contraction.
{"title":"The myosin II coiled-coil domain atomic structure in its native environment","authors":"H. Rahmani, Wen Ma, Zhongjun Hu, N. Daneshparvar, Dianne W. Taylor, J. McCammon, T. Irving, R. Edwards, K. Taylor","doi":"10.1017/S1431927621003299","DOIUrl":"https://doi.org/10.1017/S1431927621003299","url":null,"abstract":"Significance Myosin II is the molecule that produces force in muscle contraction. Unlike the myosin head, its molecular motor, no atomic resolution structure of the ∼1000-residue–long α-helical coiled-coil tail has been reported. Here, we describe the cryo-EM atomic structure of the myosin tail within a native muscle thick filament. Three differences with crystal structures of myosin tail segments were found. The myosin head arrangement apparently alters the beginning of the tail. Striated muscle myosins have four skip residues, amino acids inserted to improve the alignment of charged residue clusters. Skips 1 and 3 agree with the crystal structures. Skip 2, which is a novel structure, and Skip 4 do not. Functional consequences are suggested by the myosin tail packing. The atomic structure of the complete myosin tail within thick filaments isolated from Lethocerus indicus flight muscle is described and compared to crystal structures of recombinant, human cardiac myosin tail segments. Overall, the agreement is good with three exceptions: the proximal S2, in which the filament has heads attached but the crystal structure doesn’t, and skip regions 2 and 4. At the head–tail junction, the tail α-helices are asymmetrically structured encompassing well-defined unfolding of 12 residues for one myosin tail, ∼4 residues of the other, and different degrees of α-helix unwinding for both tail α-helices, thereby providing an atomic resolution description of coiled-coil “uncoiling” at the head–tail junction. Asymmetry is observed in the nonhelical C termini; one C-terminal segment is intercalated between ribbons of myosin tails, the other apparently terminating at Skip 4 of another myosin tail. Between skip residues, crystal and filament structures agree well. Skips 1 and 3 also agree well and show the expected α-helix unwinding and coiled-coil untwisting in response to skip residue insertion. Skips 2 and 4 are different. Skip 2 is accommodated in an unusual manner through an increase in α-helix radius and corresponding reduction in rise/residue. Skip 4 remains helical in one chain, with the other chain unfolded, apparently influenced by the acidic myosin C terminus. The atomic model may shed some light on thick filament mechanosensing and is a step in understanding the complex roles that thick filaments of all species undergo during muscle contraction.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"32 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85005039","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：