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Rxivist combines biology preprints from bioRxiv and medRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 128,741 papers from 551,614 authors.

Most downloaded biology preprints, since beginning of last month

in category molecular biology

2,931 results found. For more information, click each entry to expand.

1: Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first six months of the COVID-19 pandemic
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Posted 01 Dec 2020

Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first six months of the COVID-19 pandemic
981 downloads bioRxiv molecular biology

Joseph H. Lubin, Christine Zardecki, Elliott M. Dolan, Changpeng Lu, Zhuofan Shen, Shuchismita Dutta, John D. Westbrook, Brian P. Hudson, David S. Goodsell, Jonathan K Williams, Maria Voigt, Vidur Sarma, Lingjun Xie, Thejasvi Venkatachalam, Steven Arnold, Luz Helena Alfaro Alvarado, Kevin Catalfano, Aaliyah Khan, Erika McCarthy, Sophia Staggers, Brea Tinsley, Alan Trudeau, Jitendra Singh, Lindsey Whitmore, Helen Zheng, Matthew Benedek, Jenna Currier, Mark Dresel, Ashish Duvvuru, Britney Dyszel, Emily Fingar, Elizabeth M. Hennen, Michael Kirsch, Ali A. Khan, Charlotte Labrie-Cleary, Stephanie Laporte, Evan Lenkeit, Kailey Martin, Marilyn Orellana, Melanie Ortiz-Alvarez de la Campa, Isaac Paredes, Baleigh Wheeler, Allison Rupert, Andrew Sam, Katherine See, Santiago Soto Zapata, Paul A Craig, Bonnie L. Hall, Jennifer Jiang, Julia R. Koeppe, Stephen A. Mills, Michael J. Pikaart, Rebecca Roberts, Y Bromberg, J Steen Hoyer, Siobain Duffy, Jay Tischfield, Francesc X. Ruiz, Eddy Arnold, Jean Baum, Jesse Sandberg, Grace Brannigan, Sagar D. Khare, Stephen K. Burley

Three-dimensional structures of SARS-CoV-2 and other coronaviral proteins archived in the Protein Data Bank were used to analyze viral proteome evolution during the first six months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48,000 viral proteome sequences showed how each one of the 29 viral study proteins have undergone amino acid changes. Structural models computed for every unique sequence variant revealed that most substitutions map to protein surfaces and boundary layers with a minority affecting hydrophobic cores. Conservative changes were observed more frequently in cores versus boundary layers/surfaces. Active sites and protein-protein interfaces showed modest numbers of substitutions. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi-Gaussian distribution. Detailed results are presented for six drug discovery targets and four structural proteins comprising the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and functional interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure-based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.

2: An engineered multifunctional protein tag for advanced fluorescence imaging
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Posted 31 Jan 2021

An engineered multifunctional protein tag for advanced fluorescence imaging
958 downloads bioRxiv molecular biology

Hela Benaissa, Karim Ounoughi, Isabelle Aujard, Evelyne Fischer, Rosette Goïame, Julie Nguyen, Alison G Tebo, Chenge Li, Thomas Le Saux, Lydia Danglot, Nicolas Pietrancosta, Xavier Morin, Ludovic Jullien, Arnaud Gautier

Fluorescent reporters are essential tools in cell biology for imaging the dynamics of proteins in living cells and organisms with high spatial and temporal resolution. Chemogenetic systems made of a genetically encoded protein tag acting as an anchor for synthetic fluorophores combine the targeting selectivity of genetic tags with the advantages of synthetic fluorophores. Here, we present the directed evolution of a small 14-kDa protein tag with extended chromophore promiscuity capable of efficiently forming non-covalent fluorescent assemblies with a collection of membrane-permeant and membrane-impermeant fluorogenic chromophores displaying spectral properties spanning the entire visible spectrum. The ability to adapt the fluorescence color by choosing a different live-cell compatible fluorogenic chromophore enables to genetically encode blue, cyan, green, yellow, orange and red fluorescence with a single tag, providing an unprecedent experimental versatility. The possibility to form dark assemblies using non-fluorescent chromophores provides moreover an innovative way for switching off fluorescence on-demand in cells and organisms with high temporal resolution by chromophore replacement. Selective wash-free fluorogenic labeling of fusion proteins could be achieved with high efficiency in live cells, including delicate cultured hippocampal neurons, and in multicellular organisms, allowing high contrast imaging with various advanced microscopy techniques. The remarkable labeling efficiency and fluorescence performance allowed the multicolor imaging of dynamic processes in multicellular systems. The ability to match the spectral properties to the imaging modalities and the high photostability enabled to achieve efficient stimulated emission depletion (STED) nanoscopy of fusion proteins in live cells and live primary cultured neurons.

3: SARS-CoV-2 variants B.1.351 and B.1.1.248: Escape from therapeutic antibodies and antibodies induced by infection and vaccination
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Posted 11 Feb 2021

SARS-CoV-2 variants B.1.351 and B.1.1.248: Escape from therapeutic antibodies and antibodies induced by infection and vaccination
721 downloads bioRxiv molecular biology

Markus Hoffmann, Prerna Arora, Ruediger Gross, Alina Seidel, Bojan Hoernich, Alexander Hahn, Nadine Krueger, Luise Graichen, Heike Hofmann-Winkler, Amy Kempf, Martin Sebastian Winkler, Sebastian Schulz, Hans-Martin Jaeck, Bernd Jahrsdoerfer, Hubert Schrezenmeier, Martin Mueller, Alexander Kleger, Jan Muench, Stefan Poehlmann

The global spread of SARS-CoV-2/COVID-19 is devastating health systems and economies worldwide. Recombinant or vaccine-induced neutralizing antibodies are used to combat the COVID-19 pandemic. However, recently emerged SARS-CoV-2 variants B.1.1.7 (UK), B.1.351 (South Africa) and B.1.1.248 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies. Here, using pseudoparticles, we show that entry of UK, South Africa and Brazil variant into human cells is susceptible to blockade by entry inhibitors. In contrast, entry of the South Africa and Brazil variant was partially (Casirivimab) or fully (Bamlanivimab) resistant to antibodies used for COVID-19 treatment and was less efficiently inhibited by serum/plasma from convalescent or BNT162b2 vaccinated individuals. These results suggest that SARS-CoV-2 may escape antibody responses, which has important implications for efforts to contain the pandemic.

4: Performance comparison of reverse transcriptases for single-cell studies
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Posted 07 May 2019

Performance comparison of reverse transcriptases for single-cell studies
519 downloads bioRxiv molecular biology

Zucha Daniel, Androvic Peter, Kubista Mikael, Valihrach Lukas

Background: Recent technical advances allowing quantification of RNA from single cells are revolutionizing biology and medicine. Currently, almost all single-cell transcriptomic protocols rely on conversion of RNA to cDNA by reverse transcription (RT). However, RT is recognized as highly limiting step due to its inherent variability and suboptimal sensitivity, especially at minute amounts of RNA. Primary factor influencing RT outcome is reverse transcriptase (RTase). Recently, several new RTases with potential to decrease the loss of information during RT have been developed, but the thorough assessment of their performance is missing. Methods: We have compared the performance of 11 RTases in RT-qPCR on single-cell and 100-cell bulk templates using two priming strategies: conventional mixture of random hexamers with oligo(dT)s and reduced concentration of oligo(dT)s mimicking common single-cell RNA-Seq library preparation protocols. Based on the performance, two RTases were further tested in high-throughput single-cell experiment. Results: All RTases tested reverse transcribed low-concentration templates with high accuracy (R2 > 0.9445) but variable reproducibility (median CVRT = 40.1 %). The most pronounced differences were found in the ability to capture rare transcripts (0 - 90% reaction positivity rate) as well as in the rate of RNA conversion to cDNA (7.3 - 124.5 % absolute yield). Finally, RTase performance and reproducibility across all tested parameters were compared using Z-scores and validity of obtained results was confirmed in a single-cell model experiment. The better performing RTase provided higher positive reaction rate and expression levels and improved resolution in clustering analysis. Conclusions: We performed a comprehensive comparison of 11 RTases in low RNA concentration range and identified two best-performing enzymes (Maxima H-; SuperScript IV). We found that using better-performing enzyme (Maxima H-) over commonly-used below-average performer (SuperScript II) increases the sensitivity of single-cell experiment. Our results provide a reference for the improvement of current single-cell quantification protocols.

5: Characterization of the SARS-CoV-2 Spike in an Early Prefusion Conformation
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Posted 17 Mar 2020

Characterization of the SARS-CoV-2 Spike in an Early Prefusion Conformation
425 downloads bioRxiv molecular biology

Tingting Li, Qingbing Zheng, Hai Yu, Dinghui Wu, Wenhui Xue, Yuyun Zhang, Xiaofen Huang, Lizhi Zhou, Zhigang Zhang, Zhenghui Zha, Tingting Chen, Zhiping Wang, Jie Chen, Hui Sun, Tingting Deng, Yingbin Wang, Yixin Chen, Qinjian Zhao, Jun Zhang, Ying Gu, Shaowei Li, Ningshao Xia

Pandemic coronavirus disease 2019 (COVID-19) is caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which there are no efficacious vaccines or therapeutics that are urgently needed. We expressed three versions of spike (S) proteins—receptor binding domain (RBD), S1 subunit and S ectodomain—in insect cells. RBD appears monomer in solutions, whereas S1 and S associate into homotrimer with substantial glycosylation. The three proteins confer excellent antigenicity with six convalescent COVID-19 patient sera. Cryo-electron microscopy (cryo-EM) analyses indicate that the SARS-CoV-2 S trimer dominate in a unique conformation distinguished from the classic prefusion conformation of coronaviruses by the upper S1 region at lower position ~15 Å proximal to viral membrane. Such conformation is proposed as an early prefusion state for the SARS-CoV-2 spike that may broaden the knowledge of coronavirus and facilitate vaccine development.

6: Multi-omic rejuvenation of human cells by maturation phase transient reprogramming
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Posted 17 Jan 2021

Multi-omic rejuvenation of human cells by maturation phase transient reprogramming
405 downloads bioRxiv molecular biology

Diljeet Gill, Aled Parry, Fátima Santos, Irene Hernando-Herraez, Thomas M. Stubbs, Inês Milagre, Wolf Reik

Ageing is the gradual decline in organismal fitness that occurs over time leading to tissue dysfunction and disease. At the cellular level, ageing is associated with reduced function, altered gene expression and a perturbed epigenome. Somatic cell reprogramming, the process of converting somatic cells to induced pluripotent stem cells (iPSCs), can reverse these age-associated changes. However, during iPSC reprogramming somatic cell identity is lost, and can be difficult to reacquire as re-differentiated iPSCs often resemble foetal rather than mature adult cells. Recent work has demonstrated that the epigenome is already rejuvenated by the maturation phase of reprogramming, which suggests full iPSC reprogramming is not required to reverse ageing of somatic cells. Here we have developed the first ''maturation phase transient reprogramming'' (MPTR) method, where reprogramming factors are expressed until this rejuvenation point followed by withdrawal of their induction. Using dermal fibroblasts from middle age donors, we found that cells reacquire their fibroblast identity following MPTR, possibly as a result of persisting epigenetic memory at enhancers. Excitingly, our method substantially rejuvenated multiple cellular attributes including the transcriptome, which was rejuvenated by around 30 years as measured by a novel transcriptome clock. The epigenome, including H3K9me3 histone methylation levels and the DNA methylation ageing clock, was rejuvenated to a similar extent. The magnitude of rejuvenation instigated by MTPR is substantially greater than that achieved in previous transient reprogramming protocols. MPTR fibroblasts produced youthful levels of collagen proteins, suggesting functional rejuvenation. Overall, our work demonstrates that it is possible to separate rejuvenation from pluripotency reprogramming, which should facilitate the discovery of novel anti-ageing genes and therapies.

7: A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing
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Posted 23 Jun 2020

A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing
391 downloads bioRxiv molecular biology

Max J. Kellner, James J Ross, Jakob Schnabl, Marcus P.S. Dekens, Robert Heinen, Irina Grishkovskaya, Benedikt Bauer, Johannes Stadlmann, Luis Menéndez-Arias, Robert Fritsche-Polanz, Marianna Traugott, Tamara Seitz, Alexander Zoufaly, Manuela Födinger, Christoph Wenisch, Johannes Zuber, Vienna Covid-19 Diagnostics Initiative (VCDI), Andrea Pauli, Julius Brennecke

Global efforts to combat the Covid-19 pandemic caused by the beta coronavirus SARS-CoV-2 are currently based on RT-qPCR-based diagnostic tests. However, their high cost, moderate throughput and reliance on sophisticated equipment limit widespread implementation. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative detection method that has the potential to overcome these limitations. Here we present a rapid, robust, highly sensitive and versatile RT-LAMP based SARS-CoV-2 detection assay. Our forty-minute procedure bypasses a dedicated RNA isolation step, is insensitive to carry-over contamination, and uses a hydroxynaphthol blue (HNB)-based colorimetric readout, which allows robust SARS-CoV-2 detection from various sample types. Based on this assay we have substantially increased sensitivity and scalability by a simple nucleic acid enrichment step (bead-LAMP), established a pipette-free version for home testing (HomeDip-LAMP), and developed a version with open source enzymes that could be produced in any molecular biology setting. Our advanced, universally applicable RT-LAMP assay is a major step towards population-scale SARS-CoV-2 testing. ### Competing Interest Statement The authors have declared no competing interest.

8: Parasite co-opts a ubiquitin receptor to induce a plethora of developmental changes
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Posted 15 Feb 2021

Parasite co-opts a ubiquitin receptor to induce a plethora of developmental changes
343 downloads bioRxiv molecular biology

Weijie Huang, Allyson MacLean, Akiko Sugio, Abbas Maqbool, Macro Busscher, Shu-Ting Cho, Sophien Kamoun, Chih-Horng Kuo, Richard G.H. Immink, Saskia A Hogenhout

Obligate parasites can induce complex and substantial phenotypic changes in their hosts in ways that favour their transmission to other trophic levels. However, mechanisms underlying these changes remain largely unknown. Here, we demonstrate how SAP05 protein effectors from insect-vectored plant pathogenic phytoplasmas take control of several plant developmental processes to simultaneously prolong host lifespan and induce witch's broom-like proliferations of leaf and sterile shoots, organs colonized by phytoplasmas and vectors. SAP05 acts by mediating the concurrent degradation of SPL and GATA developmental regulators via a process that uniquely relies on hijacking the plant ubiquitin receptor RPN10 independently of substrate lysine ubiquitination. RPN10 is highly conserved among eukaryotes, but SAP05 does not bind insect vector RPN10. A two-amino-acid substitution within plant RPN10 generates a functional variant that is resistant to SAP05 activities. Therefore, one effector protein enables obligate parasitic phytoplasmas to induce a plethora of developmental phenotypes in their hosts.

9: Mechanism of dopamine binding and allosteric modulation of the human D1 dopamine receptor
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Posted 08 Feb 2021

Mechanism of dopamine binding and allosteric modulation of the human D1 dopamine receptor
324 downloads bioRxiv molecular biology

H. Eric Xu, Youwen Zhuang, Brian Krumm, Huibing Zhang, X. Edward Zhou, Yue Wang, Xi-Ping Huang, Yongfeng Liu, Xi Cheng, Yi Jiang, Hualiang Jiang, Cheng Zhang, Wei Yi, Bryan L. Roth, Yan Zhang

Dopamine is an essential neurotransmitter, which functions are mediated by five G protein-coupled receptors, dopamine D1 to D5 receptors (D1R-D5R) in mammals. Among them, D1R is the most abundantly expressed dopamine receptor in the CNS and is the central receptor mediating excitatory dopamine signaling in multiple dopaminergic pathways. Dysregulation of D1R signaling has been directly linked to Parkinson's disease (PD), schizophrenia, and drug abuse. Due to its fundamental functions in human diseases, D1R has long been the subject of intensive drug development effort toward the treatment of neuropsychiatric diseases. Here, we report the structures of D1R-Gs complex bound to endogenous agonist dopamine and synthetic agonist SKF81297, both with positive allosteric modulator LY3154207. These structures reveal the basis of dopamine recognition, the binding and potential allosteric regulation of DRD1 PAM LY3154207, and provide structural templates for design of subtype-selective D1R ligand for drug discovery targeting DRD1 for treating various CNS diseases.

10: CryoDRGN: Reconstruction of heterogeneous structures from cryo-electron micrographs using neural networks
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Posted 29 Mar 2020

CryoDRGN: Reconstruction of heterogeneous structures from cryo-electron micrographs using neural networks
321 downloads bioRxiv molecular biology

Ellen D. Zhong, Tristan Bepler, Bonnie Berger, Joseph H. Davis

Cryo-EM single-particle analysis has proven powerful in determining the structures of rigid macromolecules. However, many protein complexes are flexible and can change conformation and composition as a result of functionally-associated dynamics. Such dynamics are poorly captured by current analysis methods. Here, we present cryoDRGN, an algorithm that for the first time leverages the representation power of deep neural networks to efficiently reconstruct highly heterogeneous complexes and continuous trajectories of protein motion. We apply this tool to two synthetic and three publicly available cryo-EM datasets, and we show that cryoDRGN provides an interpretable representation of structural heterogeneity that can be used to identify discrete states as well as continuous conformational changes. This ability enables cryoDRGN to discover previously overlooked structural states and to visualize molecules in motion.

11: One-step RNA extraction for RT-qPCR detection of 2019-nCoV
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Posted 05 Apr 2020

One-step RNA extraction for RT-qPCR detection of 2019-nCoV
304 downloads bioRxiv molecular biology

Monica Sentmanat, Evguenia Kouranova, Xiaoxia Cui

The global outbreak of coronavirus disease 2019 (COVID-19) has placed an unprecedented burden on healthcare systems as the virus spread from the initial 27 reported cases in the city of Wuhan, China to a global pandemic in under three month[1]. Resources essential to monitoring virus transmission have been challenged with a demand for expanded surveillance. The CDC 2019-nCoV Real-Time Diagnostic Panel uses a real-time reverse transcription polymerase chain reaction (RT-PCR) consisting of two TaqMan probe and primer sets specific for the 2019-nCoV N gene, which codes for the nucleocapsid structural protein that encapsulates viral RNA, for the qualitative detection of 2019-nCoV viral RNA in respiratory samples. To isolate RNA from respiratory samples, the CDC lists RNA extraction kits from four manufacturers. In anticipation of a limited supply chain of RNA extraction kits and the need for test scalability, we sought to identify alternative RNA extraction methods. Here we show that direct lysis of respiratory samples can be used in place of RNA extraction kits to run the CDC 2019-nCoV Real-Time Diagnostic assay with the additional benefits of higher throughput, lower cost, faster turnaround and possibly higher sensitivity and improved safety. ### Competing Interest Statement The authors have declared no competing interest.

12: Reversal of ageing- and injury-induced vision loss by Tet-dependent epigenetic reprogramming
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Posted 31 Jul 2019

Reversal of ageing- and injury-induced vision loss by Tet-dependent epigenetic reprogramming
291 downloads bioRxiv molecular biology

Yuancheng Lu, Anitha Krishnan, Benedikt Brommer, Xiao Tian, Margarita Meer, Daniel L. Vera, Chen Wang, Qiurui Zeng, Doudou Yu, Michael S. Bonkowski, Jae-Hyun Yang, Emma M. Hoffmann, Songlin Zhou, Ekaterina Korobkina, Noah Davidsohn, Michael B. Schultz, Karolina Chwalek, Luis A. Rajman, George Church, Konrad Hochedlinger, Vadim N Gladyshev, Steve Horvath, Meredith S. Gregory-Ksander, Bruce R. Ksander, Zhigang He, David A. Sinclair

Ageing is a degenerative process leading to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise, which disrupts youthful gene expression patterns that are required for cells to function optimally and recover from damage. Changes to DNA methylation patterns over time form the basis of an 'ageing clock', but whether old individuals retain information to reset the clock and, if so, whether this would improve tissue function is not known. Of all the tissues in the body, the central nervous system (CNS) is one of the first to lose regenerative capacity. Using the eye as a model tissue, we show that expression of Oct4, Sox2, and Klf4 genes (OSK) in mice resets youthful gene expression patterns and the DNA methylation age of retinal ganglion cells, promotes axon regeneration after optic nerve crush injury, and restores vision in a mouse model of glaucoma and in normal old mice. This process, which we call recovery of information via epigenetic reprogramming or REVIVER, requires the DNA demethylases Tet1 and Tet2, indicating that DNA methylation patterns don't just indicate age, they participate in ageing. Thus, old tissues retain a faithful record of youthful epigenetic information that can be accessed for functional age reversal.

13: Perturbations in 3D genome organization can promote acquired drug resistance
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Posted 02 Feb 2021

Perturbations in 3D genome organization can promote acquired drug resistance
288 downloads bioRxiv molecular biology

Anna G Manjon, Daniel Peric-Hupkes, Ning Qing Liu, Anoek Friskes, Stacey Joosten, Hans Teunissen, Marleen Aarts, Stefan Prekovic, Wilbert Zwart, Elzo de Wit, Bas van Steensel, Rene H Medema

Acquired drug resistance is a major problem in the treatment of cancer. hTERT-immortalized, untransformed RPE-1 (RPE) cells can acquire resistance to taxol by derepressing the ABCB1 gene, encoding for the multidrug transporter P-gP. Here we have investigated how the ABCB1 gene is derepressed. We show that activation of the ABCB1 gene is associated with reduced DNA methylation, reduced H3K9 trimethylation and increased H3K27 acetylation at the ABCB1 promoter. In addition, we find that the ABCB1 locus has moved away from the nuclear lamina in the taxol-resistant cells. This raises the question which of these alterations were causal to derepression. Directly modifying DNA methylation or H3K27 methylation had neither significant effect on ABCB1 expression, nor did it promote drug resistance. In contrast, the disruption of Lamin B Receptor (LBR), a component of the nuclear lamina involved in genome organization, did promote the acquisition of a taxol-resistant phenotype in a subset of cells. Using CRISPRa-mediated gene activation, we could further substantiate a model in which disruption of lamina association renders the ABCB1 gene permissive to derepression. Based on these data we propose a model in which nuclear lamina dissociation of a repressed gene allows for its activation, implying that deregulation of the 3D genome topology could play an important role in tumor evolution and the acquisition of drug resistance.

14: The Host Interactome of Spike Expands the Tropism of SARS-CoV-2
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Posted 16 Feb 2021

The Host Interactome of Spike Expands the Tropism of SARS-CoV-2
272 downloads bioRxiv molecular biology

Tom Casimir Bamberger, Sandra Pankow, Salvador Martinez-Bartolome, Jolene Diedrich, Robin Park, John Robert Yates

The SARS-CoV-2 virus causes severe acute respiratory syndrome (COVID-19) and has rapidly created a global pandemic. Patients that survive may face a slow recovery with long lasting side effects that can afflict different organs. SARS-CoV-2 primarily infects epithelial airway cells that express the host entry receptor Angiotensin Converting Enzyme 2 (ACE2) which binds to spike protein trimers on the surface of SARS-CoV-2 virions. However, SARS-CoV-2 can spread to other tissues even though they are negative for ACE2. To gain insight into the molecular constituents that might influence SARS-CoV-2 tropism, we determined which additional host factors engage with the viral spike protein in disease-relevant human bronchial epithelial cells (16HBEo-). We found that spike recruited the extracellular proteins laminin and thrombospondin and was retained in the endoplasmatic reticulum (ER) by the proteins DJB11 and FBX2 which support re-folding or degradation of nascent proteins in the ER. Because emerging mutations of the spike protein potentially impact the virus tropism, we compared the interactome of D614 spike with that of the rapidly spreading G614 mutated spike. More D614 than G614 spike associated with the proteins UGGT1, calnexin, HSP7A and GRP78/BiP which ensure glycosylation and folding of proteins in the ER. In contrast to G614 spike, D614 spike was endoproteolytically cleaved, and the N-terminal S1 domain was degraded in the ER even though C-terminal S2-only proteoforms remained present. D614 spike also bound more laminin than G614 spike, which suggested that extracellular laminins may function as co-factors for an alternative, S2-only dependent virus entry. Because the host interactome determines whether an infection is productive, we developed a novel proteome-based cell type set enrichment analysis (pCtSEA). With pCtSEA we determined that the host interactome of the spike protein may extend the tropism of SARS-CoV-2 beyond mucous epithelia to several different cell types, including macrophages and epithelial cells in the nephron. An S2-only dependent, alternative infection of additional cell types with SARS-CoV-2 may impact vaccination strategies and may provide a molecular explanation for a severe or prolonged progression of disease in select COVID-19 patients.

15: The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells
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Posted 31 Jan 2020

The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells
272 downloads bioRxiv molecular biology

Markus Hoffmann, Hannah Kleine-Weber, Nadine Krüger, Marcel Müller, Christian Drosten, Stefan Poehlmann

The emergence of a novel, highly pathogenic coronavirus, 2019-nCoV, in China, and its rapid national and international spread pose a global health emergency. Coronaviruses use their spike proteins to select and enter target cells and insights into nCoV-2019 spike (S)-driven entry might facilitate assessment of pandemic potential and reveal therapeutic targets. Here, we demonstrate that 2019-nCoV-S uses the SARS-coronavirus receptor, ACE2, for entry and the cellular protease TMPRSS2 for 2019-nCoV-S priming. A TMPRSS2 inhibitor blocked entry and might constitute a treatment option. Finally, we show that the serum from a convalescent SARS patient neutralized 2019-nCoV-S-driven entry. Our results reveal important commonalities between 2019-nCoV and SARS-coronavirus infection, which might translate into similar transmissibility and disease pathogenesis. Moreover, they identify a target for antiviral intervention.

16: A Simple Enhancement for Gibson Isothermal Assembly
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Posted 15 Jun 2020

A Simple Enhancement for Gibson Isothermal Assembly
269 downloads bioRxiv molecular biology

Brian A Rabe, Constance L Cepko

Gibson Isothermal Assembly has become a widespread cloning method, with a multitude of advantages over traditional cut-and-paste cloning. It allows for scarless assembly of multiple fragments simultaneously and has become widely used for molecular cloning. We have found that a simple change to the formulation of the reaction mix, the addition of a single-stranded DNA binding protein, can substantially improve both the accuracy and efficiency of assembly, especially as the number of fragments being assembled increases. In addition, when creating this Enhanced Gibson Isothermal Assembly reaction mix in-house with homemade DNA ligase, the cost of the reaction can be reduced to less than $10 per milliliter. ### Competing Interest Statement The authors have declared no competing interest.

17: Tau aggregates are RNA-protein assemblies that mis-localize multiple nuclear speckle components
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Posted 27 Jan 2021

Tau aggregates are RNA-protein assemblies that mis-localize multiple nuclear speckle components
250 downloads bioRxiv molecular biology

Evan Lester, Roy Parker, Felicia K. Ooi, Nadine Bakkar, Jacob Ayers, Amanda L Woerman, Joshua R. Wheeler, Robert Bowser, George A Carlson, Stanley B. Prusiner

Tau aggregates contribute to neurodegenerative diseases including frontotemporal dementia and Alzheimers disease (AD). Although RNA promotes tau aggregation in vitro, whether tau aggregates in cells contain RNA is unknown. We demonstrate in cell culture and mouse brains that both cytosolic and nuclear tau aggregates contain RNA, with enrichment for snRNAs and snoRNAs. Nuclear tau aggregates colocalize with and alter the composition, dynamics, and organization of nuclear speckles, which are membraneless organelles involved in pre-mRNA splicing. Moreover, several nuclear speckle components, including SRRM2, mislocalize to cytosolic tau aggregates in cells, mouse brains, and patient brains with AD, frontotemporal dementia (FTD), and corticobasal degeneration (CBD). Consistent with these alterations we observe the presence of tau aggregates is sufficient to alter pre-mRNA splicing. This work identifies tau alteration of nuclear speckles as a feature of tau aggregation that may contribute to the pathology of tau aggregates.

18: A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of SARS-CoV-2
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Posted 27 Feb 2020

A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of SARS-CoV-2
241 downloads bioRxiv molecular biology

Zhen Zhao, Haodong Cui, Wenxing Song, Xiaoling Ru, Wenhua Zhou, Xuefeng Yu

The ongoing outbreak of the novel coronavirus disease 2019 (COVID-19) originating from Wuhan, China, draws worldwide concerns due to its long incubation period and strong infectivity. Although RT-PCR-based molecular diagnosis techniques are being widely applied for clinical diagnosis currently, timely and accurate diagnosis are still limited due to labour intensive and time-consuming operations of these techniques. To address the issue, herein we report the synthesis of poly (amino ester) with carboxyl groups (PC)-coated magnetic nanoparticles (pcMNPs), and the development of pcMNPs-based viral RNA extraction method for the sensitive detection of COVID-19 causing virus, the SARS-CoV-2. This method combines the lysis and binding steps into one step, and the pcMNPs-RNA complexes can be directly introduced into subsequent RT-PCR reactions. The simplified process can purify viral RNA from multiple samples within 20 min using a simple manual method or an automated high-throughput approach. By identifying two different regions (ORFlab and N gene) of viral RNA, a 10-copy sensitivity and a strong linear correlation between 10 and 100,000 copies of SARS-CoV-2 pseudovirus particles are achieved. Benefitting from the simplicity and excellent performances, this new extraction method can dramatically reduce the turn-around time and operational requirements in current molecular diagnosis of COVID-19, in particular for the early clinical diagnosis.

19: The serotonin reuptake inhibitor Fluoxetine inhibits SARS-CoV-2
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Posted 14 Jun 2020

The serotonin reuptake inhibitor Fluoxetine inhibits SARS-CoV-2
236 downloads bioRxiv molecular biology

Melissa Zimniak, Luisa Kirschner, Helen Hilpert, Jürgen Seibel, Jochen Bodem

To circumvent time-consuming clinical trials, testing whether existing drugs are effective inhibitors of SARS-CoV-2, has led to the discovery of Remdesivir. We decided to follow this path and screened approved medications off-label against SARS-CoV-2. In these screenings, Fluoxetine inhibited SARS-CoV-2 at a concentration of 0.8μg/ml significantly, and the EC50 was determined with 387ng/ml. Fluoxetine is a racemate consisting of both stereoisomers, while the S-form is the dominant serotonin reuptake inhibitor. We found that both isomers show similar activity on the virus. Fluoxetine treatment resulted in a decrease in viral protein expression. Furthermore, Fluoxetine inhibited neither Rabies virus, human respiratory syncytial virus replication nor the Human Herpesvirus 8 or Herpes simplex virus type 1 gene expression, indicating that it acts virus-specific. We see the role of Fluoxetine in the early treatment of SARS-CoV-2 infected patients of risk groups. ### Competing Interest Statement The authors have declared no competing interest.

20: Shotgun Transcriptome and Isothermal Profiling of SARS-CoV-2 Infection Reveals Unique Host Responses, Viral Diversification, and Drug Interactions
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Posted 20 Apr 2020

Shotgun Transcriptome and Isothermal Profiling of SARS-CoV-2 Infection Reveals Unique Host Responses, Viral Diversification, and Drug Interactions
229 downloads bioRxiv molecular biology

Daniel J Butler, Christopher Mozsary, Cem Meydan, David C Danko, Jonathan Foox, Joel Rosiene, Alon Shaiber, Ebrahim Afshinnekoo, Matthew MacKay, Fritz J. Sedlazeck, Nikolay A Ivanov, Maria Sierra, Diana Pohle, Michael Zietz, Undina Gisladottir, Vijendra Ramlall, Craig D Westover, Krista Ryon, Benjamin Young, Chandrima Bhattacharya, Phyllis Ruggiero, Bradley W Langhorst, Nathan Tanner, Justyna Gawrys, Dmitry Meleshko, Dong Xu, Peter A D Steel, Amos J Shemesh, Jenny Xiang, Jean Thierry-Mieg, Danielle Thierry-Mieg, Robert E. Schwartz, Angelika Iftner, Daniela Bezdan, John Sipley, Lin Cong, Arryn Craney, Priya Velu, Ari M. Melnick, Iman Hajirasouliha, Stacy M. Horner, Thomas Iftner, Mirella Salvatore, Massimo Loda, Lars F Westblade, Melissa Cushing, Sean E. Levy, Shixiu Wu, Nicholas Tatonetti, Marcin Imielinski, Hanna Rennert, Christopher Mason

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused thousands of deaths worldwide, including >18,000 in New York City (NYC) alone. The sudden emergence of this pandemic has highlighted a pressing clinical need for rapid, scalable diagnostics that can detect infection, interrogate strain evolution, and identify novel patient biomarkers. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs, plus a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, bacterial, and viral profiling. We applied both technologies across 857 SARS-CoV-2 clinical specimens and 86 NYC subway samples, providing a broad molecular portrait of the COVID-19 NYC outbreak. Our results define new features of SARS-CoV-2 evolution, nominate a novel, NYC-enriched viral subclade, reveal specific host responses in ACE, interferon, hematological, and olfaction pathways, and examine risks associated with use of ACE inhibitors and angiotensin receptor blockers. Together, these findings have immediate applications to SARS-CoV-2 diagnostics, public health monitoring, and new therapeutic targets. ### Competing Interest Statement N.T. and B.L. are employees at New England Biolabs.

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