bluuebunny/crossref_metadata_2025 huggingface dataset:
A two-point IC50method for evaluating the biochemical potency of irreversible enzyme inhibitors
Authors: Kuzmič Petr
Published: June 2020

Irreversible (covalent) enzyme inhibitors cannot be unambiguously ranked for biochemical potency by using IC50values determined at a single point in time, because the same IC50value could originate either from relatively low initial binding affinity accompanied by high chemical reactivity, or the other way around. To disambiguate the potency ranking of covalent inhibitors, here we describe a data-analytic procedure relying on two separate IC50values, determined at two different reaction times. In the case of covalent inhibitors following the two-step kinetic mechanismE+I⇌E·I→EI, the two IC50values alone can be used to estimate both the inhibition constant (Ki) as a measure of binding affinity and the inactivation rate constant (kinact) as a measure of chemical reactivity. In the case of covalent inhibitors following the one-step kinetic mechanismE+I→EI, a simple algebraic formula can be used to estimate the covalent efficiency constant (kinact/Ki) from a single experimental value of IC50. The two simplifying assumptions underlying the method are (1) zero inhibitor depletion, which implies that the inhibitor concentrations are always significantly higher than the enzyme concentration; and (2) constant reaction rate in the uninhibited control assay. The newly proposed method is validated by using a simulation study involving 64 irreversible inhibitors with covalent efficiency constants spanning seven orders of magnitude.
Circulating levels of epirubicin cause endothelial senescence while compromising metabolic activity and vascular function
Authors: Eakin Amanda J , McErlain Tamara , Burke Aileen , Eaton Amy , Tipping Nuala , Allocca Gloria , Branco Cristina M.
Published: June 2020

Anthracycline-based chemotherapy is a common treatment for cancer patients. Because it is delivered intravenously, endothelial cells are exposed first and to the highest concentrations, prior to diffusion to target cells. Not surprisingly, vascular dysfunction is a consequence of anthracycline therapy. While chemotherapy-induced endothelial damage at administration sites has been investigated, the effects of lower doses encountered by distant microvascular networks has not. The aim of this study was to investigate the impact of epirubicin, a widely used anthracycline, on healthy endothelial cells to elucidate its effects on microvascular physiology.

Here, endothelial cells were briefly exposed to low doses of epirubicin to recapitulate levels in circulation following dilution in the blood and compound half-life in circulation. Both immediate and prolonged responses to treatment were assessed to determine changes in endothelial function.

Epirubicin caused a decrease in proliferation and viability in hUVEC, with lower doses resulting in a senescent phenotype in a large proportion of cells, accompanied by a significant increase in pro-inflammatory cytokines and a significant decrease in metabolic activity. Epirubicin exposure also impaired endothelial function with delayed wound closure, reduced angiogenic potential and increased monolayer permeability downstream of VE-cadherin internalization. Primary lung endothelial cells obtained from epirubicin-treated mice similarly demonstrated reduced viability and functional impairment.In vivo, epirubicin treatment resulted in persistent reduction in lung vascular density and significantly increased infiltration of myeloid cells.

Modulation of endothelial status and inflammatory tissue microenvironment observed in response to low doses of epirubicin may predict risk for long-term secondary pathologies associated with chemotherapy.
Phylogenetic position and taxonomic rearrangement ofDavidina(Lepidoptera, Nymphalidae), an enigmatic butterfly genus new for Europe and America
Authors: Lukhtanov Vladimir A. , Dubatolov Vladimir V.
Published: June 2020

Davidina, an enigmatic butterfly genus described from China in the 19thcentury, has been long time considered a member of the family Pieridae due to its pierid-like wing pattern. In the 20th century, it was transferred to the family Satyridae (now subfamily Satyrinae of Nymphalidae) based on analysis of genitalia structure and placed next to the species-rich genusOeneis(subtribe Satyrina), being separated from the latter by supposed differences in wing venation. Here we conducted phylogenetic and taxonomic study of the subtribe Satyrina using analysis of molecular and morphological characters. We show that the genusOeneisis not monophyletic, and consists of two non-sister, genetically diverged and morphologically differentiated groups (Oeneiss.s. andProtoeneis). We also demonstrate thatDavidinais closely related toProtoeneis, not toOeneiss.s. To avoid the discovered non-monophyly and morphological heterogeneity, several species should be extracted fromOeneisand transferred to the genusDavidina. As a consequence, we conclude that the nameProtoeneisGorbunov, 2001 is congeneric withDavidinaOberthür, 1879. We also conclude thatDavidinais not a monotypic Chinese endemic genus as it has been previously supposed, but is composed of nine species and has a broad distribution area in the Holarctic region including Europe and America.
Changes in local mineral homeostasis facilitate the formation of benign and malignant testicular microcalcifications
Authors: Boisen Ida Marie , Knudsen Nadia Krarup , Nielsen John Erik , Kooij Ireen , Bagger Mathilde Louise , Kaludjerovic Jovana , O’ Shaughnessy Peter J. , Andrews Peter W. , Ide Noriko , Toft Birgitte Grønkær , Juul Anders , Mehmedbašić Arnela , Jørgensen Anne , Smith Lee B. , Norman Richard W. , Meyts Ewa Rajpert-De , Lanske Beate , Jensen Martin Blomberg
Published: June 2020

Testicular microcalcifications consist of hydroxyapatite and have been associated with an increased risk of testicular germ cell tumors (TGCTs) but are also found in benign cases such as loss-of-function variants in the phosphate-transporterSLC34A2. Here, we show that fibroblast growth factor 23 (FGF23), a regulator of phosphate homeostasis, is expressed in testicular germ cell neoplasiain situ(GCNIS), embryonal carcinoma (EC), and human embryonic stem cells. FGF23 is not glycosylated in TGCTs and therefore cleaved into a C-terminal fragment which competitively antagonizes full-length FGF23. Here,Fgf23knockout mice presented with marked calcifications in the epididymis, spermatogenic arrest, and focally germ cells expressing the osteoblast marker Osteocalcin (gene name:BGLAP, protein name: Osteocalcin). Moreover, the frequent testicular microcalcifications in mice with no functional androgen receptor and lack of circulating gonadotropins are associated with lowerSlc34a2and higherBglap/Slc34a1(protein name: NPT2a) expression compared with wild-type mice. In accordance, human testicular specimens with microcalcifications also have lowerSLC34A2and a subpopulation of germ cells express phosphate-transporter NPT2a, Osteocalcin, and RUNX2 highlighting aberrant local phosphate handling and expression of bone-specific proteins. Mineral disturbancein vitrousing calcium or phosphate treatment induced deposition of calcium-phosphate in a spermatogonial cell line and this effect was fully rescued by the mineralization-inhibitor pyrophosphate. In conclusion, testicular microcalcifications arise secondary to local alterations in mineral homeostasis, which in combination with impaired Sertoli cell function and reduced levels of mineralization-inhibitors due to high alkaline phosphatase activity in GCNIS and TGCTs, facilitate osteogenic-like differentiation of testicular cells and deposition of hydroxyapatite.
Structural basis for peptide substrate specificities of glycosyltransferase GalNAc-T2
Authors: Mahajan Sai Pooja , Srinivasan Yashes , Labonte Jason W. , DeLisa Matthew P. , Gray Jeffrey J.
Published: June 2020

The polypeptide N-acetylgalactosaminyl transferase (GalNAc-T) enzyme family initiates O-linked mucin-type glycosylation. The family constitutes 20 isozymes in humans—an unusually large number—unique to O-glycosylation. GalNAc-Ts exhibit both redundancy and finely tuned specificity for a wide range of peptide substrates. In this work, we deciphered the sequence and structural motifs that determine the peptide substrate preferences for the GalNAc-T2 isoform. Our approach involved sampling and characterization of peptide–enzyme conformations obtained from Rosetta Monte Carlo-minimization–based flexible docking. We computationally scanned 19 amino acid residues at positions −1 and +1 of an eight-residue peptide substrate, which comprised a dataset of 361 (19×19) peptides with previously characterized experimental GalNAc-T2 glycosylation efficiencies. The calculations recapitulated experimental specificity data, successfully discriminating between glycosylatable and non-glycosylatable peptides with a probability of 96.5% (ROC-AUC score), a balanced accuracy of 85.5% and a false positive rate of 7.3%. The glycosylatable peptide substrates viz. peptides with proline, serine, threonine, and alanine at the −1 position of the peptide preferentially exhibited cognate sequon-like conformations. The preference for specific residues at the −1 position of the peptide was regulated by enzyme residues R362, K363, Q364, H365 and W331, which modulate the pocket size and specific enzyme-peptide interactions. For the +1 position of the peptide, enzyme residues K281 and K363 formed gating interactions with aromatics and glutamines at the +1 position of the peptide, leading to modes of peptide-binding sub-optimal for catalysis. Overall, our work revealed enzyme features that lead to the finely tuned specificity observed for a broad range of peptide substrates for the GalNAc-T2 enzyme. We anticipate that the key sequence and structural motifs can be extended to analyze specificities of other isoforms of the GalNAc-T family and can be used to guide design of variants with tailored specificity.
GDSL-domain containing proteins mediate suberin biosynthesis and degradation, enabling developmental plasticity of the endodermis during lateral root emergence
Authors: Ursache Robertas , Vieira-Teixeira Cristovao De Jesus , Tendon Valérie Dénervaud , Gully Kay , De Bellis Damien , Schmid-Siegert Emanuel , Andersen Tonni Grube , Shekhar Vinay , Calderon Sandra , Pradervand Sylvain , Nawrath Christiane , Geldner Niko , Vermeer Joop E.M.
Published: June 2020

Roots anchor plants and deliver water and nutrients from the soil. The root endodermis provides the crucial extracellular diffusion barrier by setting up a supracellular network of lignified cell walls, called Casparian strips, supported by a subsequent formation of suberin lamellae. Whereas lignification is thought to be irreversible, formation of suberin lamellae was demonstrated to be dynamic, facilitating adaptation to different soil conditions. Plants shape their root system through the regulated formation of lateral roots emerging from within the endodermis, requiring local breaking and re-sealing of the endodermal diffusion barriers. Here, we show that differentiated endodermal cells have a distinct auxin-mediated transcriptional response that regulates cell wall remodelling. Based on this data set we identify a set of GDSL-lipases that are essential for suberin formation. Moreover, we find that another set of GDSL-lipases mediates suberin degradation, which enables the developmental plasticity of the endodermis required for normal lateral root emergence.
Identification of protein-protected mRNA fragments and structured excised intron RNAs in human plasma by TGIRT-seq peak calling
Authors: Yao Jun , Wu Douglas C. , Nottingham Ryan M. , Lambowitz Alan M.
Published: June 2020

Human plasma contains >40,000 different coding and non-coding RNAs that are potential biomarkers for human diseases. Here, we used thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) combined with peak calling to simultaneously profile all RNA biotypes in apheresis-prepared human plasma pooled from healthy individuals. Extending previous TGIRT-seq analysis, we found that human plasma contains largely fragmented mRNAs from >19,000 protein-coding genes, abundant full-length, mature tRNAs and other structured small non-coding RNAs, and less abundant tRNA fragments and mature and pre-miRNAs. Many of the mRNA fragments identified by peak calling correspond to annotated protein-binding sites and/or have stable predicted secondary structures that could afford protection from plasma nucleases. Peak calling also identified novel repeat RNAs, miRNA-sized RNAs, and putatively structured intron RNAs of potential biological, evolutionary, and biomarker significance, including a family of full-length excised introns RNAs, subsets of which correspond to mirtron pre-miRNAs or agotrons.
Sex-specific expression and DNA methylation in a species with extreme sexual dimorphism and paternal genome elimination
Authors: Bain Stevie A. , Marshall Hollie , Ross Laura
Published: June 2020

Sexual dimorphism is exhibited in many species across the tree of life with many phenotypic differences mediated by differential expression and alternative splicing of genes present in both sexes. However, the mechanisms that regulate these sex-specific expression and splicing patterns remain poorly understood. The mealybug, Planococcus citri, displays extreme sexual dimorphism and exhibits an unusual instance of sex-specific genomic imprinting, Paternal Genome Elimination (PGE), in which the paternal chromosomes in males are highly condensed and eliminated from the sperm. P. citri also has no sex chromosomes and as such both sexual dimorphism and PGE are predicted to be under epigenetic control. We recently showed that P. citri females display a highly unusual DNA methylation profile for an insect species, with the presence of promoter methylation associated with lower levels of gene expression. In this study we therefore decided to explore genome-wide differences in DNA methylation between male and female P. citri using whole genome bisulfite sequencing. We have identified extreme differences in genome-wide levels and patterns between the sexes. Males display overall higher levels of DNA methylation which manifests as more uniform low-levels across the genome. Whereas females display more targeted high levels of methylation. We suggest these unique sex-specific differences are due to chromosomal differences caused by PGE and may be linked to possible ploidy compensation. Using RNA-Seq we identified extensive sex-specific gene expression and alternative splicing. We found cis-acting DNA methylation is not directly associated with differentially expressed or differentially spliced genes, indicating a broader role for chromosome-wide trans-acting DNA methylation in this species.
Impact of vector control on effective population sizes; empirical evidence for a control-based genetic bottleneck in the tsetse flyGlossina fuscipes
Authors: Muhwezi Allan , Cunningham Lucas J. , Esterhuizen Johan , Tirados Inaki , Matovu Enock , Donnelly Martin J. , Torr Stephen J.
Published: June 2020

We investigated genetic variation at 37 newly-developed microsatellite loci in populations of the tsetse flyGlossina fuscipes fuscipescaptured from the upper and lower reaches of a single hydrographical network within an endemic Human African Trypanosomiasis focus. Our primary aim was to assess the impact of vector control using insecticide-treated baits (Tiny Targets) on genetic structure. We initially usedSTRUCTUREto delineate geographical boundaries of two stable ‘ancestral’ reference populations without any history of vector control but marked for either vector control (‘intervention’) or no control (‘non-intervention’). We then used theADMIXTUREmodel to assess genetic divergence in temporal populations collected after vector control implementation. We applied the Linkage Disequilibrium method to explicitly measure spatial and temporal changes in effective population size (Ne). We observed a significant reduction inNecoincident with vector control, whereasNeremained stable in the non-intervention area. Our empirical findings show how classical population genetics approaches detected within a short period of time, a significant genetic bottleneck associated with vector control, and opens up the possibility of using routine genomic surveillance. We have also generated a resource of new genetic markers for studies on the population genetics of tsetse at finer-scale resolution.

Funding

This work was funded through a Wellcome Trust Master’s Fellowship in Public Health and Tropical Medicine awarded to Allan Muhwezi (103268/Z/13/Z).
Scale invariance during bacterial reductive division observed by an extensive microperfusion system
Authors: Shimaya Takuro , Okura Reiko , Wakamoto Yuichi , Takeuchi Kazumasa A.
Published: June 2020

In stable environments, cell size fluctuations are thought to be governed by simple physical principles, as suggested by recent finding of scaling properties. Here we show, using E. coli, that the scaling concept also rules cell size fluctuations under time-dependent conditions, even though the distribution changes with time. We develop a microfluidic device for observing dense and large bacterial populations, under uniform and switchable conditions. Triggering bacterial reductive division by switching to non-nutritious medium, we find that the cell size distribution changes in a specific manner that keeps its normalized form unchanged; in other words, scale invariance holds. This finding is underpinned by simulations of a model based on cell growth and intracellular replication. We also formulate the problem theoretically and propose a sufficient condition for the scale invariance. Our results emphasize the importance of intrinsic cellular replication processes in this problem, suggesting different distribution trends for bacteria and eukaryotes.
Cheetah: a computational toolkit for cybergenetic control
Authors: Pedone Elisa , de Cesare Irene , Zamora-Chimal Criseida G. , Haener David , Postiglione Lorena , La Regina Antonella , Shannon Barbara , Savery Nigel J. , Grierson Claire S. , di Bernardo Mario , Gorochowski Thomas E. , Marucci Lucia
Published: June 2020

Advances in microscopy, microfluidics and optogenetics enable single-cell monitoring and environmental regulation and offer the means to control cellular phenotypes. The development of such systems is challenging and often results in bespoke setups that hinder reproducibility. To address this, we introduce Cheetah – a flexible computational toolkit that simplifies the integration of real-time microscopy analysis with algorithms for cellular control. Central to the platform is an image segmentation system based on the versatile U-Net convolutional neural network. This is supplemented with functionality to robustly count, characterise and control cells over time. We demonstrate Cheetah’s core capabilities by analysing long-term bacterial and mammalian cell growth and by dynamically controlling protein expression in mammalian cells. In all cases, Cheetah’s segmentation accuracy exceeds that of a commonly used thresholding-based method, allowing for more accurate control signals to be generated. Availability of this easy-to-use platform will make control engineering techniques more accessible and offer new ways to probe and manipulate living cells.
Balance between promiscuity and specificity in phage λ host range
Authors: Andrews Bryan , Fields Stanley
Published: June 2020

As hosts acquire resistance to viruses, viruses must overcome that resistance to re-establish infectivity, or go extinct. Despite the significant hurdles associated with adapting to a resistant host, viruses are evolutionarily successful and maintain stable coevolutionary relationships with their hosts. To investigate the factors underlying how pathogens adapt to their hosts, we performed a deep mutational scan of the region of the λ tail fiber tip protein that mediates contact with the λ host,E. coli. Phages harboring amino acid substitutions were subjected to selection for infectivity on wild typeE. coli, revealing a highly restrictive fitness landscape, in which most substitutions completely abrogate function. By comparing this lack of mutational tolerance to evolutionary diversity, we highlight a set of mutationally intolerant and diverse positions associated with host range expansion. Imposing selection for infectivity on three λ-resistant hosts, each harboring a different missense mutation in the λ receptor, reveals hundreds of adaptive variants in λ. We distinguish λ variants that confer promiscuity, a general ability to overcome host resistance, from those that drive host-specific infectivity. Both processes may be important in driving adaptation to a novel host.
DeepGANnel: Synthesis of fully annotated single molecule patch-clamp data using generative adversarial networks
Authors: Ball Sam T. M. , Celik Numan , Sayari Elaheh , Kadir Lina Abdul , O’Brien Fiona , Barrett-Jolley Richard
Published: June 2020

Development of automated analysis tools for “single ion channel” recording is hampered by the lack of available training data. For machine learning based tools, very large training sets are necessary with sample-by-sample point labelled data (e.g., 1 sample point every 100microsecond). In an experimental context, such data are labelled with human supervision, and whilst this is feasible for simple experimental analysis, it is infeasible to generate the enormous datasets that would be necessary for a big data approach using hand crafting. In this work we aimed to develop methods to generate simulated ion channel data that is free from assumptions and prior knowledge of noise and underlying hidden Markov models.

We successfully leverage generative adversarial networks (GANs) to build an end-to-end pipeline for generating an unlimited amount of labelled training data from a small annotated ion channel “seed” record, and this needs no prior knowledge of theoretical dynamical ion channel properties. Our method utilises 2D CNNs to maintain the synchronised temporal relationship between the raw and idealised record. We demonstrate the applicability of the method with 5 different data sources and show authenticity with t-SNE and UMAP projection comparisons between real and synthetic data. The model would be easily extendable to other time series data requiring parallel labelling, such as labelled ECG signals or raw nanopore sequencing data.
Dramatic shape changes occur as Cytochrome c folds
Authors: Kirmizialtin Serdal , Pitici Felicia , Cardenas Alfredo E , Elber Ron , Thirumalai D.
Published: June 2020

Extensive experimental studies on the folding of Cytochrome c (Cyt c) make this small protein an ideal target for atomic detailed simulations for the purposes of quantitatively characterizing the structural transitions and the associated time scales for folding to the native state from an ensemble of unfolded states. We use previously generated atomically detailed folding trajectories by the Stochastic Difference Equation in Length (SDEL) to calculate the time-dependent changes in the Small Angle X-ray scattering (SAXS) profiles. Excellent agreement is obtained between experiments and simulations for the time dependent SAXS spectra, allowing us to identify the structures of the folding intermediates, which shows that Cyt c reaches the native state by a sequential folding mechanism. Using the ensembles of structures along the folding pathways we show that compaction and the sphericity of Cyt c change dramatically from the prolate ellipsoid shape in the unfolded state to the spherical native state. Our data, which provides unprecedented quantitative agreement with all aspects of time-resolved SAXS experiments, shows that hydrophobic collapse and amide group protection coincide on the 100 microseconds time scale, which is in accord with ultrafast Hydrogen/Deuterium exchange studies. Based on these results we propose that compaction of polypeptide chains, accompanied by dramatic shape changes, is a universal characteristic of globular proteins, regardless of the underlying folding mechanism.
Cold tolerance of mountain stoneflies (Plecoptera: Nemouridae) from the high Rocky Mountains
Authors: Hotaling Scott , Shah Alisha A. , Dillon Michael E. , Giersch J. Joseph , Tronstad Lusha M. , Finn Debra S. , Woods H. Arthur , Kelley Joanna L.
Published: June 2020

How aquatic insects cope with cold temperatures is poorly understood. This is particularly true for high-elevation species that often experience a seasonal risk of freezing. In the Rocky Mountains, nemourid stoneflies (Plecoptera: Nemouridae) are a major component of mountain stream biodiversity and are typically found in streams fed by glaciers and snowfields, which due to climate change, are rapidly receding. Predicting the effects of climate change on mountain stoneflies is difficult because their thermal physiology is largely unknown. We investigated cold tolerance of several alpine stoneflies (Lednia tumana, Lednia tetonica, and Zapada spp.) from the Rocky Mountains, USA. We measured the supercooling point (SCP) and tolerance to ice enclosure of late-instar nymphs collected from a range of thermal regimes. SCPs varied among species and populations, with the lowest SCP measured for nymphs from an alpine pond, which is much more likely to freeze solid in winter than flowing streams. We also show that L. tumana cannot survive being enclosed in ice, even for short periods of time (less than three hours) at relatively mild temperatures (−0.5 °C). Our results indicate that high-elevation stoneflies at greater risk of freezing may have correspondingly lower SCPs, and despite their common association with glacial meltwater, they appear to be living near their lower thermal limits.
Abundance and nuclear antigen reactivity of intestinal and fecal Immunoglobulin A in lupus-prone mice at younger ages correlate with the onset of eventual systemic autoimmunity
Authors: Sun Wei , Gudi Radhika , Johnson Benjamin M. , Vasu Chenthamarakshan
Published: June 2020

Our recent studies, using (SWRxNZB)F1 (SNF1) mice, showed a potential contribution of the gut microbiota and pro-inflammatory immune responses of the gut mucosa to systemic autoimmunity in lupus. Here, using this mouse model, we determined the abundance and the nAg reactivity of IgA antibody produced in the intestine under lupus susceptibility. Intestinal lymphoid tissues from SNF1 mice, females particularly, showed significantly higher frequencies of nAg (dsDNA and nucleohistone) reactive IgA producing B cells compared to B6 females. Most importantly, younger age fecal IgA -abundance and - nAg reactivity of lupus-prone mice showed a positive correlation with eventual systemic autoimmunity and proteinuria onset. Depletion of gut microbiota in SNF1 mice resulted in the diminished production of IgA in the intestine and the nAg reactivity of these antibodies. Overall, these observations show that fecal IgA features, nuclear antigen reactivity particularly, at preclinical stages/in at-risk subjects could be predictive of autoimmune progression.
Cortical Organoids Model Early Brain Development Disrupted by 16p11.2 Copy Number Variants in Autism
Authors: Urresti Jorge , Zhang Pan , Moran-Losada Patricia , Yu Nam-Kyung , Negraes Priscilla D. , Trujillo Cleber A. , Antaki Danny , Amar Megha , Chau Kevin , Pramod Akula Bala , Diedrich Jolene , Tejwani Leon , Romero Sarah , Sebat Jonathan , Yates John R. , Muotri Alysson R. , Iakoucheva Lilia M.
Published: June 2020

Reciprocal deletion and duplication of 16p11.2 region is the most common copy number variation (CNV) associated with Autism Spectrum Disorders. We generated cortical organoids from skin fibroblasts of patients with 16p11.2 CNV to investigate impacted neurodevelopmental processes. We show that organoid size recapitulates macrocephaly and microcephaly phenotypes observed in the patients with 16p11.2 deletions and duplications. The CNV has mirror-opposite effect on neuronal maturation, proliferation, and synapse number, in concordance with its effect on brain growth in humans. We demonstrate that 16p11.2 CNV alters the ratio of neurons to neural progenitors in organoids during early neurogenesis, with excess of neurons and depletion of neural progenitors observed in deletions, and mirror phenotypes in duplications. Transcriptomic and proteomic profiling revealed multiple dysregulated pathways, including defects in neuron migration. Inhibition of activity of the small GTPase RhoA rescued migration deficits. This study provides insights into potential neurobiological mechanisms behind the 16p11.2 CNV during neocortical development.
Using nucleocapsid proteins to investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses
Authors: Schuster Noah
Published: June 2020

An initial outbreak of coronavirus disease 2019 (COVID-19) in China has resulted in a massive global pandemic causing well over 16,500,000 cases and 650,000 deaths worldwide. The virus responsible, SARS-CoV-2, has been found to possess a very close association with Bat-CoV RaTG13 and Pangolin-CoV MP789. The nucleocapsid protein can serve as a decent model for determining phylogenetic, evolutionary, and structural relationships between coronaviruses. Therefore, this study uses the nucleocapsid gene and protein to further investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses. Sequence and phylogenetic analyses have revealed the nucleocapsid gene and protein in SARS-CoV-2 are both closely related to those found in Bat-CoV RaTG13 and Pangolin-CoV MP789. Evidence of recombination was detected within the N gene, along with the presence of a double amino acid insertion found in the N-terminal region. Homology modeling for the N-Terminal Domain revealed similar structures but distinct electrostatic surfaces and topological variations in the β-hairpin that likely reflect specific adaptive functions. In respect to SARS-CoV-2, two amino acids (S37 and A267) were found to exist only in its N protein, along with an extended β-hairpin that bends towards the nucleotide binding site. Collectively, this study strengthens the relationship among SARS-CoV-2, Bat-CoV RaTG13, and Pangolin-CoV MP789, providing additional insights into the structure and adaptive nature of the nucleocapsid protein found in these coronaviruses. Furthermore, these data will enhance our understanding of the complete history behind SARS-CoV-2 and help assist in antiviral and vaccine development.
A PCNA-K164R mutation impinges on origin activation and mitotic DNA synthesis
Authors: Leung Wendy , Baxley Ryan M. , Thakar Tanay , Chang Ya-Chu , Rogers Colette B. , Wang Liangjun , Durrett Wesley , Tella Anika , Moldovan George-Lucian , Shima Naoko , Bielinsky Anja-Katrin
Published: June 2020

Ubiquitination of the replication clamp proliferating cell nuclear antigen (PCNA) at the conserved residue lysine 164 (K164) occurs during normal S phase progression and increases after DNA damage induced replication stress. PCNA-K164 ubiquitination is critical for Okazaki fragment maturation and the activation of DNA damage tolerance pathways. Moreover, ubiquitinated PCNA operates in a fork protection pathway parallel to BRCA-RAD51. Whether PCNA ubiquitination regulates other genome maintenance mechanisms is unclear. UtilizingPCNAK164Rcells generated by CRISPR-Cas9, we demonstrate that this mutation causes DNA replication defects that impact origin activation.PCNAK164Rcells accumulate single-stranded DNA gaps during replication that persist throughout mitosis due to compromised mitotic DNA synthesis (MiDAS). We uncover a novel role for PCNA-K164 ubiquitination in regulating FANCD2 to initiate MiDAS. Persistent gaps hence interfere with MCM2-7 double hexamer loading in the subsequent G1 phase. Our findings demonstrate that the impact of PCNAK164-Ubis not limited to S/G2 phases but extends to mitosis and G1 phase.

PCNA-K164 ubiquitination promotes DNA gap filling during S/G2 phases of the cell cycle. This study identifies a novel role for K164 ubiquitination in replication dynamics and mitotic DNA synthesis and thus provides new insight into the players involved in counteracting under-replication.
Different tertiary interactions create the same important 3-D features in a divergent flavivirus xrRNA
Authors: Jones Rachel A. , Steckelberg Anna-Lena , Szucs Matthew J. , Akiyama Benjamin M. , Vicens Quentin , Kieft Jeffrey S.
Published: June 2020

During infection by a flavivirus (FV), cells accumulate noncoding subgenomic flavivirus RNAs (sfRNAs) that interfere with several antiviral pathways. These sfRNAs are formed by structured RNA elements in the 3′ untranslated region (UTR) of the viral genomic RNA, which block the progression of host cell exoribonucleases that have targeted the viral RNA for destruction. Previous work on these exoribonuclease-resistant RNAs (xrRNAs) from mosquito-borne FVs revealed a specific 3-dimensional fold with a unique topology in which a ring-like structure protectively encircles the 5′ end of the xrRNA. Conserved nucleotides make specific tertiary interactions that support this fold. Examination of more divergent FVs reveals differences in their 3′ UTR sequences, raising the question of whether they contain xrRNAs and if so, how they fold. To answer this, we demonstrated the presence of an authentic xrRNA in the 3′ UTR of the Tamana Bat Virus (TABV) and solved its structure by x-ray crystallography. The structure reveals conserved features from previously characterized xrRNAs, but in the TABV version these features are created through a novel set of tertiary interactions not previously seen in xrRNAs. This includes two important A-C interactions, four distinct backbone kinks, several ordered Mg2+ ions, and a C+-G-C base triple. The discovery that the same overall architecture can be achieved by very different sequences and interactions in distantly related flaviviruses provides insight into the diversity of this type of RNA and will inform searches for undiscovered xrRNAs in viruses and beyond.
Dense time-course gene expression profiling of the Drosophila melanogaster innate immune response
Authors: Schlamp Florencia , Delbare Sofie Y. N. , Early Angela M. , Wells Martin T. , Basu Sumanta , Clark Andrew G.
Published: June 2020

Background

Immune responses need to be initiated rapidly, and maintained as needed, to prevent establishment and growth of infections. At the same time, resources need to be balanced with other physiological processes. On the level of transcription, studies have shown that this balancing act is reflected in tight control of the initiation kinetics and shutdown dynamics of specific immune genes.

Results

To investigate genome-wide expression dynamics and trade-offs after infection at a high temporal resolution, we performed an RNA-seq time course on D. melanogaster with 20 time points post Imd stimulation. A combination of methods, including spline fitting, cluster analysis, and Granger causality inference, allowed detailed dissection of expression profiles, lead-lag interactions, and functional annotation of genes through guilt-by-association. We identified Imd-responsive genes and co-expressed, less well characterized genes, with an immediate-early response and sustained up-regulation up to five days after stimulation. In contrast, stress response and Toll-responsive genes, among which were Bomanins, demonstrated early and transient responses. We further observed a strong trade-off with metabolic genes, which strikingly recovered to pre-infection levels before the immune response was fully resolved.

Conclusions

This high-dimensional dataset enabled the comprehensive study of immune response dynamics through the parallel application of multiple temporal data analysis methods. The well annotated data set should also serve as a useful resource for further investigation of the D. melanogaster innate immune response, and for the development of methods for analysis of a post-stress transcriptional response time-series at whole-genome scale.
Nanotrap®particles improve detection of SARS-CoV-2 for pooled sample methods, extraction-free saliva methods, and extraction-free transport medium methods
Authors: Barclay RA , Akhrymuk I , Patnaik A , Callahan V , Lehman C , Andersen P , Barbero R , Barksdale S , Dunlap R , Goldfarb D , Jones-Roe T , Kelly R , Kim B , Miao S , Munns A , Munns D , Patel S , Porter E , Ramsey R , Sahoo S , Swahn O , Warsh J , Kehn-Hall K , Lepene B
Published: June 2020

Here we present a rapid and versatile method for capturing and concentrating SARS-CoV-2 from transport medium and saliva using affinity-capture magnetic hydrogel particles. We demonstrate that the method concentrates virus prior to RNA extraction, thus significantly improving detection of the virus using a real-time RT-PCR assay across a range of viral titers, from 100 to 1,000,000 viral copies/mL; in particular, detection of virus in low viral load samples is enhanced when using the method coupled with the IDT 2019-nCoV CDC EUA Kit. This method is compatible with commercially available nucleic acid extraction kits, as well with a simple heat and detergent method. Using transport medium diagnostic remnant samples that previously had been tested for SARS-CoV-2 using either the Abbott RealTime SARS-CoV-2 EUA Test (n=14) or the Cepheid Xpert Xpress SARS-CoV-2 EUA Test (n=35), we demonstrate that our method not only correctly identifies all positive samples (n = 17) but also significantly improves detection of the virus in low viral load samples. The average improvement in cycle threshold (Ct) value as measured with the IDT 2019-nCoV CDC EUA Kit was 3.1; n = 10. Finally, to demonstrate that the method could potentially be used to enable pooled testing, we spiked infectious virus or a confirmed positive diagnostic remnant sample into 5 mL and 10 mL of negative transport medium and observed significant improvement in the detection of the virus from those larger sample volumes.
Cytoplasmic Microtubule Organizing Centers Regulate Meiotic Spindle Positioning in Mouse Oocyte
Authors: Vasquez Daniela Londono , Rodriguez-Lukey Katherine , Behura Susanta K. , Balboula Ahmed Z.
Published: June 2020

During oocyte meiosis, migration of the spindle and its positioning must be tightly regulated to ensure elimination of the polar bodies and provide developmentally competent euploid eggs. Although the role of F-actin in regulating these critical processes has been studied extensively, little is known whether microtubules (MTs) participate in regulating these processes. Here, we characterize a pool of MTOCs in the oocyte that does not contribute to spindle assembly but instead remains free in the cytoplasm during metaphase I (metaphase cytoplasmic MTOCs; mcMTOCs). In contrast to spindle pole MTOCs, which primarily originate from the perinuclear region in prophase I, the mcMTOCs are found near the cortex of the oocyte. At nuclear envelope breakdown, they exhibit robust nucleation of MTs, which diminishes during polar body extrusion before returning robustly during metaphase II. The asymmetric positioning of the mcMTOCs provides the spindle with a MT-based anchor line to the cortex opposite the site of polar body extrusion. Depletion of mcMTOCs, by laser ablation, or manipulating their numbers, through autophagy inhibition, revealed that the mcMTOCs are required to regulate the timely migration and positioning of the spindle in meiosis. We discuss how forces exerted by F-actin in mediating movement of the spindle to the oocyte cortex are balanced by MT-mediated forces from the mcMTOCs to ensure spindle positioning and timely spindle migration.
Assessment of the use and quick preparation of saliva for rapid microbiological diagnosis of COVID-19
Authors: Fernández-Pittol Mariana , Hurtado Juan Carlos , Moreno-García Estela , Rubio Elisa , Navarro Mireia , Valiente Marta , Peiró Aida , Capón Alicia , Seijas Nuria , Martínez Miguel J. , Casals-Pascual Climent , Vila Jordi
Published: June 2020

The objective of this study was to assess the performance of direct real time RT-PCR detection of SARS-CoV-2 in heated saliva samples, avoiding the RNA isolation step. Oropharyngeal and nasopharyngeal swabs together with saliva samples were obtained from 51 patients clinically diagnosed as potentially having COVID-19. Two different methods were compared: 1. RNA was extracted from 500 μl of sample using a MagNA Pure Compact Instrument with an elution volume of 50μl and 2. 700µL of saliva were heat-inactivated at 96°C for 15 minutes, and directly subjected to RT-PCR. One step real time RT-PCR was performed using 5 μl of extracted RNA or directly from 5 μl of heated sample. RT-PCR was performed targeting the SARS-CoV-2 envelope (E) gene region. Diagnostic performance was assessed using the results of the RT-PCR from nasopharyngeal and oropharyngeal swabs as the gold standard. The overall sensitivity, specificity, positive and negative predictive values were 81.08%, 92.86%, 96.77% and 65.00%, respectively when RNA extraction was included in the protocol with saliva, whereas sensitivity, specificity, positive and negative predictive values were 83.78%, 92.86%, 68.42% and 96.88%, respectively, for the heat-inactivation protocol. However, when the analysis was performed exclusively on saliva samples with a limited time from the onset of symptoms (<9 days, N=28), these values were 90%, 87.5%, 44% and 98.75% for the heat-inactivation protocol. The study showed that RT-PCR can be performed using saliva in an RNA extraction free protocol, showing good sensitivity and specificity.
Blood parameters measured on admission as predictors of outcome for COVID-19; a prospective UK cohort study
Authors: Arnold DT , Attwood M , Barratt S , Elvers K , Morley A , Noel A , MacGowan A , Maskell NA , Hamilton F
Published: June 2020

Introduction

COVID-19 has an unpredictable clinical course so prognostic biomarkers would be invaluable when triaging patients on admission to hospital. Many biomarkers have been suggested using large observational datasets but sample timing is crucial to ensure prognostic relevance. The DISCOVER study prospectively recruited patients with COVID-19 admitted to a UK hospital and analysed a panel of putative prognostic biomarkers on the admission blood sample to identify markers of poor outcome.

Methods

Consecutive patients admitted to hospital with proven or clinicoradiological suspected COVID-19 were recruited. Admission bloods were extracted from the clinical laboratory. A panel of biomarkers (IL-6, suPAR, KL-6, Troponin, Ferritin, LDH, BNP, Procalcitonin) were performed in addition to routinely performed markers (CRP, neutrophils, lymphocytes, neutrophil:lymphocyte ratio). Age, NEWS score and CURB-65 were included as comparators. All biomarkers were tested in logistic regression against a composite outcome of non-invasive ventilation, intensive care admission, or death, with Area Under the Curve (AUC) figures calculated.

Results

155 patients had 28-day outcomes at the time of analysis. CRP (AUC 0.51, CI:0.40-0.62), lymphocyte count (AUC 0.62, CI:0.51-0.72), and other routine markers did not predict the primary outcome. IL-6 (AUC: 0.78,0.65-0.89) and suPAR (AUC 0.77, CI: 0.66-0.85) showed some promise, but simple clinical features alone such as NEWS score (AUC: 0.74, 0.64-0.83) or age (AUC: 0.70, 0.61-0.78) performed nearly as well.

Discussion

Admission blood biomarkers have only moderate predictive value for predicting COVID-19 outcomes, while simple clinical features such as age and NEWS score outperform many biomarkers. IL-6 and suPAR had the best performance, and further studies should validate these biomarkers in a prospective fashion.
SARS-CoV-2 has been circulating in northern Italy since December 2019: evidence from environmental monitoring
Authors: La Rosa Giuseppina , Mancini Pamela , Ferraro Giusy Bonanno , Veneri Carolina , Iaconelli Marcello , Bonadonna Lucia , Lucentini Luca , Suffredini Elisabetta
Published: June 2020

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease COVID-19, a public health emergency worldwide, and Italy is among the world’s first and most severely affected countries. The first autochthonous Italian case of COVID-19 was documented on February 21. We investigated the possibility that SARS-CoV-2 emerged in Italy earlier than that date, by analysing 40 composite influent wastewater samples collected - in the framework of other wastewater-based epidemiology projects - between October 2019 and February 2020 from five wastewater treatment plants (WTPs) in three cities and regions in northern Italy (Milan/Lombardy, Turin/Piedmont and Bologna/Emilia Romagna). Twenty-four additional samples collected in the same WTPs between September 2018 and June 2019 were included as blank samples. Viral concentration was performed according to the standard World Health Organization procedure for poliovirus sewage surveillance. Molecular analysis was undertaken with both nested RT-PCR and real-rime RT-PCR assays. A total of 15 positive samples were confirmed by both methods. Of these, 8 were collected before the first autochthonous Italian case. The earliest dates back to 18 December 2019 in Milan and Turin and 29 January 2020 in Bologna. Samples collected in January and February in the three cities were also positive.

Our results demonstrate that SARS-CoV-2 was already circulating in northern Italy at the end of 2019. Moreover, it was circulating in different geographic regions simultaneously, which changes our previous understanding of the geographical circulation of the virus in Italy. Our study highlights once again the importance of environmental surveillance as an early warning system, to monitor the levels of virus circulating in the population and identify outbreaks even before cases are notified to the healthcare system.