bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultra-rapid detection of SARS-CoV-2 in public workspace environments
Authors: Yaren Ozlem , McCarter Jacquelyn , Phadke Nikhil , Bradley Kevin M. , Overton Benjamin , Yang Zunyi , Ranade Shatakshi , Patil Kunal , Bangale Rishikesh , Benner Steven A.
Published: October 2020

Managing the pandemic caused by SARS-CoV-2 requires new capabilities in testing, including the possibility of identifying, in minutes, infected individuals as they enter spaces where they must congregate in a functioning society, including workspaces, schools, points of entry, and commercial business establishments. Here, the only useful tests (a) require no sample transport, (b) require minimal sample manipulation, (c) can be performed by unlicensed individuals, (d) return results on the spot in much less than one hour, and (e) cost no more than a few dollars. The sensitivity need not be as high as normally required by the FDA for screening asymptomatic carriers (as few as 10 virions per sample), as these viral loads are almost certainly not high enough for an individual to present a risk for forward infection. This allows tests specifically useful for this pandemic to trade-off unneeded sensitivity for necessary speed, simplicity, and frugality. In some studies, it was shown that viral load that creates forward-infection risk may exceed 105 virions per milliliter, easily within the sensitivity of an RNA amplification architecture, but unattainable by antibody-based architectures that simply target viral antigens. Here, we describe such a test based on a displaceable probe loop amplification architecture.
The Challenge of Forecasting Demand of Medical Resources and Supplies During a Pandemic: A Comparative Evaluation of Three Surge Calculators for COVID-19
Authors: Kamar A.A. , Maalouf N. , Hitti E. , Eid G. El , Isma’eel H. , Elhajj I. H.
Published: October 2020

Ever since the World Health Organization (WHO) declared the new coronavirus disease 2019 (COVID-19) as a pandemic, there has been a public health debate concerning medical resources and supplies including hospital beds, intensive care units (ICU), ventilators, and Protective Personal Equipment (PPE). Forecasting COVID-19 dissemination has played a key role in informing healthcare professionals and governments on how to manage overburdened healthcare systems. However, forecasting during the pandemic remained challenging and sometimes highly controversial. Here, we highlight this challenge by performing a comparative evaluation for the estimations obtained from three COVID-19 surge calculators under different social distancing approaches, taking Lebanon as a case study. Despite discrepancies in estimations, the three surge calculators used herein agree that there will be a relative shortage in the capacity of medical resources and a significant surge in PPE demand as the social distancing policy is removed. Our results underscore the importance of implementing containment interventions including social distancing in alleviating the demand for medical care during the COVID-19 pandemic in the absence of any medication or vaccine. It is said that “All models are wrong, but some are useful,” in this paper we highlight that it is even more useful to employ several models.
Vagus Nerve and Stomach Synucleinopathy in Parkinson’s Disease, Incidental Lewy Body Disease and Normal Elderly Subjects: Evidence Against the “Body-First” Hypothesis
Authors: Beach Thomas G. , Adler Charles H. , Sue Lucia I. , Shill Holly A. , Driver-Dunckley Erika , Mehta Shyamal H. , Intorcia Anthony J. , Glass Michael J. , Walker Jessica E. , Arce Richard , Nelson Courtney M. , Serrano Geidy E.
Published: October 2020

Braak and others have proposed that Lewy-type α-synucleinopathy (aSyn) in Parkinson’s disease (PD) may arise from an exogenous pathogen that passes across the gastric mucosa and then is retrogradely transported up the vagus nerve to the medulla. We tested this “body-first” hypothesis by immunohistochemically staining stomach and vagus nerve tissue from an autopsy series of 111 normal elderly subjects (no brain aSyn), 33 with incidental Lewy body disease (ILBD) (brain aSyn without clinical parkinsonism or dementia) and 53 with PD. Median disease duration for the PD group was 13 years. Vagus nerve samples were taken adjacent to the carotid artery in the neck. Stomach samples were taken from the gastric body, midway along the greater curvature. Formalin-fixed paraffin-embedded sections were immunohistochemically stained for α-synuclein phosphorylated at serine-129. In the vagus nerve none of the 111 normal subjects had aSyn in the vagus, while 12/26 ILBD (46%) and 32/36 PD (89%) subjects were aSyn-positive. In the stomach none of the 102 normal subjects had aSyn while 5/30 (17%) ILBD and 42/52 (81%) of PD subjects were aSyn-positive. As there was no aSyn in the vagus nerve or stomach of subjects without brain aSyn, these results support initiation of aSyn in the brain. The presence of aSyn in the vagus nerve and stomach of a subset of ILBD cases indicates that progression of synucleinopathy to the peripheral nervous system may occur at preclinical stages of Lewy body disease.
Intravital Deep-Tumor Single-Beam 2-, 3- and 4-Photon Microscopy
Authors: Bakker Gert-Jan , Weischer Sarah , Heidelin Judith , Andresen Volker , Beutler Marcus , Friedl Peter
Published: October 2020

Three-photon excitation has recently been introduced to perform intravital microscopy in deep, previously inaccessible layers of the brain. The applicability of deep-tissue three-photon excitation in more heterogeneously structured, dense tissue types remains, however, unclear. Here we show that in tumors and bone, high-pulse-energy low-duty-cycle infrared excitation near 1300 and 1700 nm enables two-up to fourfold increased tissue penetration compared to conventional 2-photon excitation. Using a single laser line, simultaneous 2-, 3- and 4-photon processes are effectively induced, enabling the simultaneous detection of blue to far-red fluorescence together with second and third harmonic generation. This enables subcellular resolution at power densities in the focus that are not phototoxic to live cells and without color aberration. Thus, infrared high-pulse-energy low-duty-cycle excitation advances deep intravital microscopy in strongly scattering tissue and, in a single scan, delivers rich multi-parameter datasets from cells and complex organ structures.
Efficient Multiplex Genome Editing Tools identified by Protoplast Technology inPhalaenopsis
Authors: Xia Keke , Zhang Dengwei , Liu Guangyu , Xu Xiaojing , Yang Yong , Zhang Guo-Qiang , Sun Hai-Xi , Gu Ying
Published: October 2020

Phalaenopsisorchids are popular ornamental plants worldwide. The application of the efficient multiplex genome editing tools inPhalaenopsis, will greatly accelerate the development of orchid gene function and breeding research. In this study, we establish a fast and convenientPhalaenopsisprotoplast platform for the identification of functional genome editing tools. Two multiplex genome editing tools, PTG-Cas9 (PTG, polycistronic tRNA gRNA) system and PTGm-Cas9 (PTG-Cas9 system with modified sgRNA structure) system are designed to editPDSgene of commercialPhalaenopsisST166 at four target sites. We find that both PTG-Cas9 and PTGm-Cas9 system are functional inPhalaenopsis, and the PTGm-Cas9 system with modified sgRNA has a higher editing efficiency than PTG-Cas9 system. Further, we design another multiplex genome editing tool, termed as DPII-Cpf1 system (dual Pol II promoter to drive the expression of Cpf1 endonuclease and crRNA), to editPDSgene ofPhalaenopsisat four target sites likewise. All the four targets are efficiently edited by DPII-Cpf1 system, and the total mutation rate is about 3 times higher than that of PTGm-Cas9 system. Taken together, using thePhalaenopsisprotoplast platform, we successfully establish two efficient multiplex genome editing tools forPhalaenopsisresearch, PTGm-Cas9 and DPII-Cpf1. The multiplex genome editing tools established in this study have great application potentials in efficiently constructing large-scale knockout mutant libraries of orchid and speeding up orchid precise breeding.
Genomic structure predicts metabolite dynamics in microbial communities
Authors: Gowda Karna , Ping Derek , Mani Madhav , Kuehn Seppe
Published: October 2020

The metabolic function of microbial communities has played a defining role in the evolution and persistence of life on Earth, driving redox reactions that form the basis of global biogeochemical cycles. Community metabolism emerges from a hierarchy of processes including gene expression, ecological interactions, and environmental factors. In wild communities, gene content is correlated with environmental context, but predicting metabolic dynamics from genomic structure remains elusive. Here we show, for the process of denitrification, that community metabolism is predictable from the genes each member of the community possesses. Machine learning reveals a sparse and generalizable mapping from gene content to metabolite dynamics across a genomically-diverse library of bacteria. A consumer-resource model correctly predicts community metabolism from single-strain phenotypes. Our results demonstrate that the conserved impacts of metabolic genes can predict community function, enabling the prediction of metabolite dynamics from metagenomes, designing denitrifying communities, and discovering how genome evolution impacts metabolism.
Characterization and analysis of the transcriptome inArapaima gigasusing multi-tissue RNA-sequencing
Authors: Martins Danilo L. , Campos Leonardo R. S. , Ribeiro-dos-Santos André M. , Coelho Ana Carolina M. F. , Dantas Renata L. , Sobrinho Pitágoras A. A. , Sakamoto Tetsu , Vidal Amanda F. , Vinasco-Sandoval Glória T. , Assumpção Paulo P. , Santos Ândrea K. C. R. , Dalmolin Rodrigo J. S. , de Souza Sandro J. , Santos Sidney , de Souza Jorge E. S.
Published: October 2020

Arapaima gigasis a giant bony tongue air-breathing fish, and a promising species for aquaculture due to its particular features. However, there is still a lack of information on its biology and few transcriptome studies are available. Our aim was to characterize the transcriptome of arapaima in order to shed light on molecular networks contributing to its unique traits. Through RNA-sequencing, we generated a transcriptome from eight tissues (brain, pituitary, heart, muscle, kidney, lung, ovary, and testis) collected from arapaima adults specimens. Using a genome-guided strategy associated with homologous protein evidence, 57,706 transcripts were assembled, which aligned to 23,353 high confidence protein-coding genes. The analysis revealed a global view of expression patterns, as well as it allowed us to identify tissue-specific gene clusters, transcription factors within the clusters, and to compare expression patterns between male and female. These analyses has generated tissue-specific and sex-biased transcriptome profiles, which will be helpful to understand its molecular biology, evolution, and also guide future functional studies of the arapaima.
Spatially resolved transcriptome profiles of mammalian kidneys illustrate the molecular complexity of functional nephron segments, cell-to-cell interactions and genetic variants
Authors: Raghubar Arti M. , Pham Duy T. , Tan Xiao , Grice Laura F. , Crawford Joanna , Lam Pui Yeng , Andersen Stacey B. , Yoon Sohye , Ng Monica S.Y. , Teoh Siok Min , Holland Samuel E. , Stewart Anne , Francis Leo , Combes Alexander N. , Kassianos Andrew J. , Healy Helen , Nguyen Quan , Mallett Andrew J.
Published: October 2020

Understanding the molecular mechanisms underlying mammalian kidney function requires transcriptome profiling of the interplay between cells comprising nephron segments. Traditional transcriptomics requires cell dissociation, resulting in loss of the spatial context of gene expression within native tissue. To address this problem, we performed spatial transcriptomics (ST) to retain the spatial context of the transcriptome in human and mouse kidneys. The generated ST data allowed spatially resolved differential gene expression analysis, spatial identification of functional nephron segments, cell-to-cell interaction analysis, and chronic kidney disease-associated genetic variant calling. Novel ST thus provides an opportunity to enhance kidney diagnostics and knowledge, by retaining the spatial context of gene expression within intact tissue.
GM-CSF differentiation of human monocytes stabilizes macrophage state via oxidative signaling
Authors: Itoh Christopher Y , Gunnarsson Cal , Babunovic Gregory H , Nibasumba Armel , Akilimali Ngomu Akeem , Wadsworth Marc H , Hughes Travis K , Solomon Sydney L , Hie Brian , Berger Bonnie , Shalek Alex K , Fortune Sarah M , Bryson Bryan D
Published: October 2020

Macrophages are central mediators of immunity that integrate diverse signals derived from differentiation cues, tissue location, and disease. Controlling macrophage state and function is an appealing therapeutic objective across many diseases including cancer, atherosclerosis, and tuberculosis. Despite the growing appreciation for thein vivocomplexity of macrophage state, existingin vitromodels of human monocyte-derived macrophages have used a limited number of individual perturbations to explore the complex phenotypic space that macrophages can occupy. Here, we leverage a tiered differentiation, activation, and stimulation strategy to generate libraries ofin vitromonocyte-derived macrophages and examine thein vitrostate space of macrophage function using high-dimensional technologies. Our tiered experimental approach further revealed a striking relationship between GM-CSF differentiation and IL-10 production. Cells that were differentiated with GM-CSF produced very low or undetectable levels of IL-10 independent of activation or stimulation condition. To nominate candidate regulators of this IL-10 response, we leverage unbiased single-cell mRNA sequencing to identify transcriptional networks associated with GM-CSF-derived cells. Using these data, we identify oxidative signaling pathways as upregulated in GM-CSF derived cells and demonstrate that scavenging of oxidative radicals can enhance IL-10 production in these cells. Collectively, these data underscore the complexity of monocyte-derived macrophage state over time and highlight a dominant role for GM-CSF in tuning macrophage inflammatory phenotype, metabolic state, and plasticity.
Chimeric single α-helical domains as rigid fusion protein connections for protein nanotechnology and structural biology
Authors: Collu Gabriella , Bierig Tobias , Krebs Anna-Sophia , Engilberge Sylvain , Varma Niveditha , Guixà-González Ramon , Deupi Xavier , Olieric Vincent , Poghosyan Emiliya , Benoit Roger M.
Published: October 2020

Chimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible, which makes them unsuitable as structural building blocks. Here we show that the ER/K motif, a single α-helical domain (SAH)1, can be seamlessly fused2to terminal helices of proteins, forming an extended and partially free-standing rigid helix. Through the intrinsic stability of the SAH, two domains can be connected with a defined distance and orientation. We designed three constructs termed YFPnano, T4Lnano, and MoStoNano, and we show that a single SAH allows the connection of two separate structural domains with sufficient rigidity to form ordered crystals. The analysis of experimentally determined structures and molecular dynamics simulations reveals a certain degree of plasticity in the connections that allows the adaptation to crystal contact opportunities. Our data show that SAHs can be stably integrated into designed structural elements, enabling new possibilities for protein nanotechnology, for example to improve the exposure of epitopes on nanoparticles (structural vaccinology), to engineer crystal contacts with minimal impact in construct flexibility (for the study of protein dynamics), and to design novel biomaterials.
iGIST - a kinetic bioassay for pertussis toxin based on its effect on inhibitory GPCR signaling
Authors: Paramonov Valeriy M. , Sahlgren Cecilia , Rivero-Müller Adolfo , Pulliainen Arto T.
Published: October 2020

Detection of pertussis toxin (PTX) activity is instrumental for the development and manufacturing of pertussis vaccines. These quality and safety measures require annually thousands of mice. Here, we describe iGIST (Interference inGαi-mediatedSignalTransduction) - an animal-free kinetic bioassay for detection of PTX by measuring its effect on inhibitory G protein-coupled receptor (GPCR) signaling. PTX ADP-ribosylates inhibitory α-subunits of the heterotrimeric G proteins, thereby perturbing the inhibitory GPCR signaling. iGIST is based on HEK293 cells co-expressing a somatostatin receptor 2 (SSTR2), which is an inhibitory GPCR controllable by a high affinity agonist octreotide, and a luminescent 3’5’-cyclic adenosine monophosphate (cAMP) probe. iGIST has a low sensitivity threshold in picogram/ml range of PTX, surpassing by 100-fold in a parallel analysis the currently usedin vitroend-point technique to detect PTX, the cluster formation assay (CFA) in Chinese hamster ovary cells. iGIST also detects PTX in complex samples, i.e. a commercial PTX- toxoid containing pertussis vaccine that was spiked with an active PTX. iGIST has an objective digital readout and is observer-independent, offering prospects for automation. iGIST emerges as a promising animal-free alternative to detect PTX activity in the development and manufacturing of pertussis vaccines. iGIST is also expected to facilitate basic PTX research, including identification and characterization of novel compounds interfering with PTX.
Positive selection inhibits plasmid coexistence in bacterial genomes
Authors: Carrilero Laura , Kottara Anastasia , Guymer David , Harrison Ellie , Hall James P. J. , Brockhurst Michael A.
Published: October 2020

Plasmids play an important role in bacterial evolution by transferring niche adaptive functional genes between lineages, thus driving genomic diversification. Bacterial genomes commonly contain multiple, coexisting plasmid replicons (i.e., plasmid coinfection), which could fuel adaptation by increasing the range of gene functions available to selection and allowing their recombination. However, plasmid coinfection is difficult to explain because the acquisition of plasmids typically incurs high fitness costs for the host cell. Here, we show that plasmid coinfection was stably maintained without positive selection for plasmid-encoded gene functions and was associated with compensatory evolution to reduce fitness costs. By contrast, with positive selection, plasmid coinfection was unstable despite compensatory evolution. Positive selection discriminated between differential fitness benefits of functionally redundant plasmid replicons, retaining only the more beneficial plasmid. These data suggest that while the efficiency of negative selection against plasmid fitness costs declines over time due to compensatory evolution, positive selection to maximise plasmid-derived fitness benefits remains efficient. Our findings help to explain the forces structuring bacterial genomes: Coexistence of multiple plasmids in a genome is likely to require either rare positive selection in nature or non-redundancy of accessory gene functions among coinfecting plasmids.
Geospatial Analysis of Individual and Community-Level Socioeconomic Factors Impacting SARS-CoV-2 Prevalence and Outcomes
Authors: Cromer Sara J. , Lakhani Chirag M. , Wexler Deborah J , Burnett-Bowie Sherri-Ann M. , Udler Miriam , Patel Chirag J.
Published: October 2020

Background

The SARS-CoV-2 pandemic has disproportionately affected racial and ethnic minority communities across the United States. We sought to disentangle individual and census tract-level sociodemographic and economic factors associated with these disparities.

Methods and Findings

All adults tested for SARS-CoV-2 between February 1 and June 21, 2020 were geocoded to a census tract based on their address; hospital employees and individuals with invalid addresses were excluded. Individual (age, sex, race/ethnicity, preferred language, insurance) and census tract-level (demographics, insurance, income, education, employment, occupation, household crowding and occupancy, built home environment, and transportation) variables were analyzed using linear mixed models predicting infection, hospitalization, and death from SARS-CoV-2.

Among 57,865 individuals, per capita testing rates, individual (older age, male sex, non-White race, non-English preferred language, and non-private insurance), and census tract-level (increased population density, higher household occupancy, and lower education) measures were associated with likelihood of infection. Among those infected, individual age, sex, race, language, and insurance, and census tract-level measures of lower education, more multi-family homes, and extreme household crowding were associated with increased likelihood of hospitalization, while higher per capita testing rates were associated with decreased likelihood. Only individual-level variables (older age, male sex, Medicare insurance) were associated with increased mortality among those hospitalized.

Conclusions

This study of the first wave of the SARS-CoV-2 pandemic in a major U.S. city presents the cascade of outcomes following SARS-CoV-2 infection within a large, multi-ethnic cohort. SARS-CoV-2 infection and hospitalization rates, but not death rates among those hospitalized, are related to census tract-level socioeconomic characteristics including lower educational attainment and higher household crowding and occupancy, but not neighborhood measures of race, independent of individual factors.
Estimation of novel coronavirus (covid-19) reproduction number and case fatality rate: a systematic review and meta-analysis
Authors: Ahammed Tanvir , Anjum Aniqua , Rahman Mohammad Meshbahur , Haider Najmul , Kock Richard , Uddin Md. Jamal
Published: October 2020

Understanding the transmission dynamics and the severity of the novel coronavirus disease 2019 (COVID-19) informs public health interventions, surveillance, and planning. Two important parameters, the basic reproduction number (R0) and case fatality rate (CFR) of COVID-19, help in this understanding process. The objective of this study was to estimate the R0 and CFR of COVID-19 and assess whether the parameters vary in different regions of the world. We carried out a systematic review to retrieve the published estimates of the R0 and the CFR in articles from international databases between 1st January and 31st August 2020. Random-effect models and Forest plots were implemented to evaluate the mean effect size of the R0 and the CFR. Furthermore, the R0 and CFR of the studies were quantified based on geographic location, the tests/thousand population, and the median population age of the countries where studies were conducted. The I2 statistic and the Cochran’s Q test were applied to assess statistical heterogeneity among the selected studies. Forty-five studies involving R0 and thirty-four studies involving CFR were included. The pooled estimation of the R0 was 2.69 (95% CI: 2.40, 2.98), and that of the CFR was 2.67 (2.25, 3.13). The CFR in different regions of the world varied significantly, from 2.51 (2.12, 2.95) in Asia to 7.11 (6.38, 7.91) in Africa. We observed higher mean CFR values for the countries with lower tests (3.15 vs. 2.16) and greater median population age (3.13 vs. 2.27). However, the R0 did not vary significantly in different regions of the world. An R0 of 2.69 and CFR of 2.67 indicate the severity of the COVID-19. Although R0 and CFR may vary over time, space, and demographics, we recommend considering these figures in control and prevention measures.
Accessing mitochondrial protein import in living cells by protein microinjection
Authors: Bogorodskiy Andrey , Okhrimenko Ivan , Maslov Ivan , Maliar Nina , Burkatovskiy Dmitry , von Ameln Florian , Schulga Alexey , Jakobs Philipp , Altschmied Joachim , Haendeler Judith , Katranidis Alexandros , Mishin Alexey , Gordeliy Valentin , Büldt Georg , Voos Wolfgang , Gensch Thomas , Borshchevskiy Valentin
Published: October 2020

Mitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment. We developed a gentle microinjection procedure for fluorescent reporter proteins allowing a direct non-invasive study of protein transport in living cells. As a proof of principle, we visualized potential-dependent protein import into mitochondria inside intact cells in real-time. We validated that our approach does not distort mitochondrial morphology and preserves the endogenous expression system as well as mitochondrial protein translocation machinery. We observed that a release of nascent polypeptides chains from actively translating cellular ribosomes by puromycin strongly increased the import rate of the microinjected preprotein. This suggests that a substantial amount of mitochondrial translocase complexes were involved in co-translational protein import of endogenously expressed preproteins. Our protein microinjection method opens new possibilities to study the role of mitochondrial protein import in cell models of various pathological conditions as well as aging processes.
DET1-mediated COP1 regulation avoids HY5 activity over second-site targets to tune plant photomorphogenesis
Authors: Cañibano Esther , Bourbousse Clara , Garcia-Leon Marta , Wolff Lea , Garcia-Baudino Camila , Barneche Fredy , Rubio Vicente , Fonseca Sandra
Published: October 2020

DE-ETIOLATED1 (DET1) is a negative regulator of plant photomorphogenesis acting as a component of the C3D complex, which can further associate to CULLIN4 to form a CRL4C3DE3 ubiquitin ligase. CRL4C3Dis thought to act together with CRL4COP1SPAubiquitin ligase, to promote the ubiquitin-mediated degradation of the master regulatory transcription factor ELONGATED HYPOCOTYL5 (HY5), thereby controlling photomorphogenic gene regulatory networks. Yet, functional links between COP1 and DET1 have long remained elusive. Here, upon mass spectrometry identification of DET1 and COP1-associated proteins, we providein vivoevidence that DET1 associates with COP1 to promote its destabilization, a process necessary to dampen HY5 protein abundance. By regulating HY5 over-accumulation, DET1 is critical to avoid its association to second-site loci, including many PIF3 target genes. Accordingly, excessive HY5 levels result in an increased HY5 repressive activity and are sufficient to triggerfusca-like phenotypes otherwise observed typically inCOP1andCOP9signalosome mutant seedlings. This study therefore identifies that DET1-mediated regulation of COP1 stability tunes down HY5 cistrome and avoids hyper-photomorphogenic responses that might compromise plant viability.
A human monoclonal antibody targeting a conserved pocket in the SARS-CoV-2 receptor-binding domain core
Authors: Fedry Juliette , Hurdiss Daniel L. , Wang Chunyan , Li Wentao , Obal Gonzalo , Drulyte Ieva , Howes Stuart C. , van Kuppeveld Frank J.M. , Förster Friedrich , Bosch Berend-Jan
Published: October 2020

SARS-CoV-2 has caused a global outbreak of severe respiratory disease (COVID-19), leading to an unprecedented public health crisis. To date, there has been over thirty-three million diagnosed infections, and over one million deaths. No vaccine or targeted therapeutics are currently available. We previously identified a human monoclonal antibody, 47D11, capable of cross-neutralising SARS-CoV-2 and the related 2002/2003 SARS-CoV in vitro, and preventing SARS-CoV-2 induced pneumonia in a hamster model. Here we present the structural basis of its neutralization mechanism. We describe cryo-EM structures of trimeric SARS-CoV and SARS-CoV-2 spike ectodomains in complex with the 47D11 Fab. These data reveal that 47D11 binds specifically to the closed conformation of the receptor binding domain, distal to the ACE2 binding site. The CDRL3 stabilises the N343 glycan in an upright conformation, exposing a conserved and mutationally constrained hydrophobic pocket, into which the CDRH3 loop inserts two aromatic residues. Interestingly, 47D11 preferentially selects for the partially open conformation of the SARS-CoV-2 spike, suggesting that it could be used effectively in combination with other antibodies that target the exposed receptor-binding motif. Taken together, these results expose a cryptic site of vulnerability on the SARS-CoV-2 RBD and provide a structural roadmap for the development of 47D11 as a prophylactic or post-exposure therapy for COVID-19.
The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells
Authors: Peacock Thomas P. , Goldhill Daniel H. , Zhou Jie , Baillon Laury , Frise Rebecca , Swann Olivia C. , Kugathasan Ruthiran , Penn Rebecca , Brown Jonathan C. , Sanchez-David Raul Y. , Braga Luca , Williamson Maia Kavanagh , Hassard Jack A. , Staller Ecco , Hanley Brian , Osborn Michael , Giacca Mauro , Davidson Andrew D. , Matthews David A. , Barclay Wendy S.
Published: October 2020

SARS-CoV-2 enters cells via its spike glycoprotein which must be cleaved sequentially at the S1/S2, then the S2’ cleavage sites (CS) to mediate membrane fusion. SARS-CoV-2 has a unique polybasic insertion at the S1/S2 CS, which we demonstrate can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture adapted SARS-CoV-2 virus with a S1/S2 deletion, we show that the polybasic insertion is selected for in lung cells and primary human airway epithelial cultures but selected against in Vero E6, a cell line used for passaging SARS-CoV-2. We find this selective advantage depends on expression of the cell surface protease, TMPRSS2, that allows virus entry independent of endosomes thus avoiding antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin CS was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals. Thus, the polybasic CS is a key determinant for efficient SARS-CoV-2 transmission.
Lipid-bound ApoE3 self-assemble into elliptical disc-shaped particles
Authors: Larsen Andreas Haahr , Johansen Nicolai Tidemand , Gajhede Michael , Arleth Lise , Midtgaard Søren Roi
Published: October 2020

Apolipoproteins are vital to lipid metabolism and cholesterol transport in the human body. Here we present a structural study of the lipid-bound particles formed by ApoE3 in a full-length and a truncated version. The particles are formed with, respectively, POPC and DMPC and investigated by small-angle X-ray scattering and negative stain electron microscopy. We find that lipid-bound ApoE3 particles are elliptical, disc-shaped particles composed of a central lipid bilayer encircled by two amphipathic ApoE3 proteins. We went on to investigate a truncated form of ApoE3 containing only residue 80 to 255 (ApoE380-255), which is the central helical repeat segment of ApoE3. The lipid-bound ApoE380-255particles are found to have the same morphology as the particles with full-length ApoE3. However, they are larger, and form more heterogeneous discoidal structures with four proteins per particle. This behavior is in contrast to ApoA1 where the highly similar helical repeat domain determines the size and stoichiometry of the formed particles both in the case of full-length and truncated ApoA1. Our data hence points towards different mechanisms for lipid bilayer structural modulation by ApoA1 and ApoE3 due to different roles of the non-repeat segments.
Direct shoot regeneration from cotyledon, leaf and root ofCitrus jambhiriLush
Authors: Sharma Priyanka , Roy Bidhan
Published: October 2020

Citrus jambhiri(Rough lemon) is popularly preferred for rootstock for cultivated species ofCitrus. Tissue culture is an appreciable technique for mass-multiplication of plant propagules. In this communication direct regeneration of plantlets ofCitrus jambhiriLush. were obtained from cotyledons, roots and leaves. Most of the cotyledon (96%) enlarged on medium supplemented with 50 mg/L of casein hydrolysate. Few of those enlarged cotyledons responded to direct regeneration of shoots. Maximum shoot per responded cotyledon was 32. Conversely, the health of the plantlets were poor with semi-cylindrical leaves. Most of them dried on maintenance medium or on rooting medium ad died. Plantlets regenerated on medium supplemented with IAA in combination with IBA were healthy and they established on maintenance medium and rooted on rooting medium. Direct regeneration was also obtained from leaf on MS medium supplemented with 0.50 mg/L of dicamba. Our finding concluded that tissue culture tools may be used for direct regeneration of plantlets from different explants ofC. jambhirito obtained true-to-type plant propagules.
Use of non-invasive intracranial pressure pulse waveform to monitor patients with End-Stage Renal Disease (ESRD) Intracranial pressure in patients with ESRD
Authors: Rickli Cristiane , Cosmoski Lais Daiene , dos Santos Fábio André , Frigieri Gustavo Henrique , Rabelo Nicollas Nunes , Schuinski Adriana Menegat , Mascarenhas Sérgio , Vellosa José Carlos Rebuglio
Published: October 2020

End-stage renal disease (ESRD) is treated mainly by hemodialysis, however, hemodialysis is associated with frequent complications, some of them involve the increased intracranial pressure. In this context, monitoring the intracranial pressure of these patients may lead to a better understanding of how intracranial pressure morphology varies with hemodialysis. This study aimed to follow-up patients with ESRD by monitoring intracranial pressure before and after hemodialysis sessions using a noninvasive method. We followed-up 42 patients with ESRD in hemodialysis, for six months. Noninvasive intracranial pressure monitoring data were obtained through analysis of intracranial pressure waveform morphology, this information was uploaded to Brain4care®cloud algorithm for analysis. The cloud automatically sends a report containing intracranial pressure parameters. In total, 4881 data points were collected during the six months of follow-up. The intracranial pressure parameters (time to peak and P2/P1 ratio) were significantly higher in predialysis when compared to postdialysis for the three weekly sessions and throughout the follow-up period (p<0.01) data showed general improvement in brain compliance after the hemodialysis session. Furthermore, intracranial pressure parameters were significantly higher in the first weekly hemodialysis session (p<0.05). In conclusion, there were significant differences between pre and postdialysis intracranial pressure in patients with ESRD on hemodialysis. Additionally, the pattern of the intracranial pressure alterations was consistent over time suggesting that hemodialysis can improve time to peak and P2/P1 ratio which may reflect in brain compliance.
Genetic interactions among ADAMTS metalloproteases and basement membrane molecules in cell migration inCaenorhabditis elegans
Authors: Imanishi Ayaka , Aoki Yuma , Kakehi Masaki , Mori Shunsuke , Takano Tomomi , Kubota Yukihiko , Kim Hon-Song , Shibata Yukimasa , Nishiwaki Kiyoji
Published: October 2020

During development of theCaenorhabditis elegansgonad, the gonadal leader cells, called distal tip cells (DTCs), migrate in a U-shaped pattern to form the U-shaped gonad arms. The ADAMTS (adisintegrinandmetalloprotease withthrombospondin motifs) family metalloproteases MIG-17 and GON-1 are required for correct DTC migration. Mutations inmig-17result in misshapen gonads due to the misdirected DTC migration, and mutations ingon-1result in shortened and swollen gonads due to the premature termination of DTC migration. Although the phenotypes shown bymig-17andgon-1mutants are very different from one another, mutations that result in amino acid substitutions in the same basement membrane protein genes,emb-9/collagen IV a1, let-2/collagen IV a2andfbl-1/fibulin-1, were identified as genetic suppressors ofmig-17andgon-1mutants. To understand the roles shared by these two proteases, we examined the effects of themig-17suppressors ongon-1and the effects of thegon-1suppressors and enhancers onmig-17gonadal defects. Some of theemb-9, let-2andfbl-1mutations suppressed bothmig-17andgon-1, whereas others acted only onmig-17orgon-1. These results suggest thatmig-17andgon-1have their specific functions as well as functions commonly shared between them for gonad formation. The levels of collagen IV accumulation in the DTC basement membrane were significantly higher in thegon-1mutants as compared with wild type and were reduced to the wild-type levels when combined with suppressor mutations, but not with enhancer mutations, suggesting that the ability to reduce collagen IV levels is important forgon-1suppression.
Social Network Analysis of the Genealogy of Strawberry: Retracing the Wild Roots of Heirloom and Modern Cultivars
Authors: Pincot Dominique D.A. , Ledda Mirko , Feldmann Mitchell J. , Hardigan Michael A. , Poorten Thomas J. , Runcie Daniel E. , Heffelfinger Christopher , Dellaporta Stephen L. , Cole Glenn S. , Knapp Steven J.
Published: October 2020

The widely recounted story of the origin of cultivated strawberry (Fragaria×ananassa) oversimplifies the complex interspecific hybrid ancestry of the highly admixed populations from which heirloom and modern cultivars have emerged. To develop deeper insights into the three century long domestication history of strawberry, we reconstructed the genealogy as deeply as possible—pedigree records were assembled for 8,851 individuals, including 2,656 cultivars developed since 1775. The parents of individuals with unverified or missing pedigree records were accurately identified by applying exclusion analysis to array-genotyped single nucleotide polymorphisms. We identified 187 wild octoploid and 1,171F.×ananassafounders in the genealogy, from the earliest hybrids to modern cultivars. The pedigree networks for cultivated strawberry are exceedingly complex labyrinths of ancestral interconnections formed by diverse hybrid ancestry, directional selection, migration, admixture, bottlenecks, overlapping generations, and recurrent hybridization with common ancestors that have unequally contributed allelic diversity to heirloom and modern cultivars. Fifteen to 333 ancestors were predicted to have transmitted 90% of the alleles found in country-, region-, and continent-specific populations. Using parent-offspring edges in the global pedigree network, we found that selection cycle lengths over the last 200 years of breeding have been extraordinarily long (16.0-16.9 years/generation) but decreased to a present-day range of 6.0-10.0 years/generation. Our analyses uncovered conspicuous differences in the ancestry and structure of North American and European populations and shed light on forces that have shaped phenotypic diversity inF.×ananassa.
Effects of sheared chromatin length on ChIP-seq quality and sensitivity
Authors: Keller Cheryl A. , Heuston Elisabeth F. , Giardine Belinda , Long Maria R. , Miller Amber , Wixom Alexander Q. , Anderson Stacie M. , Bodine David M. , Hardison Ross C.
Published: October 2020

Chromatin immunoprecipitation followed by massively parallel, high throughput sequencing (ChIP-seq) is the method of choice for identifying, on a genome-wide scale, the segments of DNA bound by specific transcription factors (TFs) or in chromatin with particular histone modifications. However, the quality of ChIP-seq datasets vary over a wide range, with a substantial fraction being of intermediate to poor quality. Such experimental variability can lead to many false positives or false negatives, impairing the ability to interpret the data. Thus, it is important to discern and control the factors that contribute to variation in ChIP-seq. In this study, we focus on the sonication step to produce sheared chromatin, a variable controllable by the user and applicable to all ChIP-seq protocols. We systematically varied the amount of shearing of fixed chromatin from a mouse erythroid cell line, carefully measured the distribution of resultant fragment lengths using the Agilent Bioanalzyer 2100, and then immunoprecipitated these batches of chromatin using highly specific antibodies against either TAL1 or CTCF. We found that the level of sonication, which was affected by both the number of sonication cycles, as well as the starting cell number, had a pronounced impact on the quality of resulting ChIP-seq signals. Specifically, over-sonication led to degradation of quality (e.g. increased background and reduction in signal), while the impact of under-sonication of chromatin differed between the two transcription factors, leading to the loss of sites occupied by TAL1 but not those bound by CTCF. We leveraged these findings to produce a set of CTCF ChIP-seq datasets in primary hematopoietic progenitor cells, including several rare cell types. Together, these results suggest that the amount of sonication is a key variable in success of ChIP-seq experiments, and that carefully monitoring the level of chromatin sonication is one way to improve ChIP-seq quality and reproducibility, which in turn facilitates low input ChIP-seq in rare cell types.
Expression of a human cDNA in moss results in spliced mRNAs and fragmentary protein isoforms
Authors: Top Oguz , Milferstaedt Stella W. L. , van Gessel Nico , Hoernstein Sebastian N. W. , Özdemir Bugra , Decker Eva L. , Reski Ralf
Published: October 2020

Production of biopharmaceuticals relies on the expression of mammalian cDNAs in host organisms. Here we show that the expression of a human cDNA in the mossPhyscomitrella patensgenerates the expected full-length and four additional transcripts due to unexpected splicing. This mRNA splicing results in non-functional protein isoforms, cellular misallocation of the proteins and low product yields. We integrated these results together with the results of our analysis of all 32,926 protein-encodingP. patensgenes and their 87,533 annotated transcripts in a web application, physCO, for automatized codon-optimization. A thus optimized cDNA results in about eleven times more protein, which correctly localizes to the ER. An analysis of codon preferences of different production hosts suggests that similar effects also occur in non-plant hosts. We anticipate that the use of our methodology will prevent so far undetected mRNA heterosplicing resulting in maximized functional protein amounts for basic biology and biotechnology.
The genotype-phenotype landscape of an allosteric protein
Authors: Tack Drew S. , Tonner Peter D. , Pressman Abe , Olson Nathanael D. , Levy Sasha F. , Romantseva Eugenia F. , Alperovich Nina , Vasilyeva Olga , Ross David
Published: October 2020

Allostery is a fundamental biophysical mechanism that underlies cellular sensing, signaling, and metabolism. Yet a quantitative understanding of allosteric genotype-phenotype relationships remains elusive. Here we report the large-scale measurement of the genotype-phenotype landscape for an allosteric protein: thelacrepressor fromEscherichia coli, LacI. Using a method that combines long-read and short-read DNA sequencing, we quantitatively measure the dose-response curves for nearly 105variants of the LacI genetic sensor. The resulting data provide a quantitative map of the effect of amino acid substitutions on LacI allostery and reveal systematic sequence-structure-function relationships. We find that in many cases, allosteric phenotypes can be quantitatively predicted with additive or neural-network models, but unpredictable changes also occur. For example, we were surprised to discover a new band-stop phenotype that challenges conventional models of allostery and that emerges from combinations of nearly silent amino acid substitutions.
Harmonicity aids hearing in noise
Authors: McPherson Malinda J. , Grace River C. , McDermott Josh H.
Published: October 2020

Hearing in noise is a core problem in audition, and a challenge for hearing-impaired listeners, yet the underlying mechanisms are poorly understood. We explored whether harmonic frequency relations, a signature property of many communication sounds, aid hearing in noise for normal hearing listeners. We measured detection thresholds in noise for tones and speech synthesized to have harmonic or inharmonic spectra. Harmonic signals were consistently easier to detect than otherwise identical inharmonic signals. Harmonicity also improved discrimination of sounds in noise. The largest benefits were observed for two-note up-down “pitch” discrimination and melodic contour discrimination, both of which could be performed equally well with harmonic and inharmonic tones in quiet, but which showed large harmonic advantages in noise. The results show that harmonicity facilitates hearing in noise, plausibly by providing a noise-robust pitch cue that aids detection and discrimination.

Significance statement

Noise is ubiquitous, and being able to hear in noise is critical to real-world behavior. We report that hearing in noise is aided by sensitivity to the harmonic frequency relations that occur in vocal communication signals and music: harmonic sounds are easier to hear in noise than inharmonic sounds. This effect was present in both musicians and non-musicians and for synthetic as well as natural sounds, suggesting a role in everyday hearing.
Growth-dependent signals drive an increase in early G1 cyclin concentration to link cell cycle entry with cell growth
Authors: Sommer Robert A. , DeWitt Jerry T. , Tan Raymond , Kellogg Douglas R.
Published: October 2020

Entry into the cell cycle occurs only when sufficient growth has occurred. In budding yeast, the cyclin Cln3 initiates cell cycle entry by inactivating a transcriptional repressor called Whi5. Growth-dependent changes in the concentrations of Cln3 or Whi5 have been proposed to link cell cycle entry to cell growth. However, there are conflicting reports regarding the behavior and roles of Cln3 and Whi5. Here, we found no evidence that changes in the concentration of Whi5 play a major role in controlling cell cycle entry. Rather, the data suggest that cell growth triggers cell cycle entry by driving an increase in the concentration of Cln3. We further found that accumulation of Cln3 is dependent upon homologs of mammalian SGK kinases that play roles in control of cell growth and size. Together, the data are consistent with models in which Cln3 serves as the crucial link between the cell cycle and signals that control cell growth and size.
Early growth response 2 (EGR2) is a novel regulator of the senescence program
Authors: Tyler Eleanor J. , del Arroyo Ana Gutierrez , Wallis Ryan , Hughes Bethany , Garbe James C. , Stampfer Martha R. , Koh Jim , Lowe Robert , Philpott Michael P , Bishop Cleo L.
Published: October 2020

Senescence, a state of stable growth arrest, plays an important role in ageing and age-related diseasesin vivo. Although the INK4/ARF locus is known to be essential for senescence programs, the key regulators drivingp16andARFtranscription remain largely underexplored. Using siRNA screening for modulators of the p16/pRB and ARF/p53/p21 pathways in deeply senescent human mammary epithelial cells (DS HMECs) and fibroblasts (DS HMFs), we identified EGR2 as a novel regulator of senescence. EGR2 expression is up-regulated during senescence and its ablation by siRNA in DS HMECs and HMFs transiently reverses the senescent phenotype. We demonstrate that EGR2 activates theARFandp16promoters and directly binds to theARFpromoter. Loss of EGR2 downregulates p16 levels and increases the pool of p16- p21- ‘reversed’ cells in the population. Moreover, EGR2 overexpression is sufficient to induce senescence. Our data suggest that EGR2 is a regulator of the p16/pRB and direct transcriptional activator of the ARF/p53/p21 pathways in senescence and a novel marker of senescence.