bluuebunny/crossref_metadata_2025 huggingface dataset:
Deciphering a marine bone degrading microbiome reveals a complex community effort
Authors: Borchert Erik , García-Moyano Antonio , Sanchez-Carrillo Sergio , Dahlgren Thomas G. , Slaby Beate M. , Bjerga Gro Elin Kjæreng , Ferrer Manuel , Franzenburg Sören , Hentschel Ute
Published: May 2020

The marine bone biome is a complex assemblage of macro- and microorganisms, however the enzymatic repertoire to access bone-derived nutrients remains unknown. The bone matrix is a composite material made up mainly of organic collagen and inorganic hydroxyapatite. We conducted field experiments to study microbial assemblages that can use organic bone components as nutrient source. Bovine and turkey bones were deposited at 69 m depth in a Norwegian fjord (Byfjorden, Bergen). Metagenomic sequence analysis was used to assess the functional potential of microbial assemblages from bone surface and the bone eating worm Osedax mucofloris, which is a frequent colonizer of whale falls and known to degrade bone. The bone microbiome displayed a surprising taxonomic diversity revealed by the examination of 59 high-quality metagenome assembled genomes from at least 23 bacterial families. Over 700 genes encoding enzymes from twelve relevant enzymatic families pertaining to collagenases, peptidases, glycosidases putatively involved in bone degradation were identified. Metagenome assembled genomes (MAGs) of the class Bacteroidia contained the most diverse gene repertoires. We postulate that demineralization of inorganic bone components is achieved by a timely succession of a closed sulfur biogeochemical cycle between sulfur-oxidizing and sulfur-reducing bacteria, causing a drop in pH and subsequent enzymatic processing of organic components in the bone surface communities. An unusually large and novel collagen utilization gene cluster was retrieved from one genome belonging to the gammaproteobacterial genus Colwellia.

Importance

Bones are an underexploited, yet potentially profitable feedstock for biotechnological advances and value chains, due to the sheer amounts of residues produced by the modern meat and poultry processing industry. In this metagenomic study we decipher the microbial pathways and enzymes that we postulate to be involved in bone degradation marine environment. We herein demonstrate the interplay between different bacterial community members, each supplying different enzymatic functions with the potential to cover an array of reactions relating to the degradation of bone matrix components. We identify and describe a novel gene cluster for collagen utilization, which is a key function in this unique environment. We propose that the interplay between the different microbial taxa is necessary to achieve the complex task of bone degradation in the marine environment.
Fear Conditioning Potentiates the Hippocampal CA1 Commissural Pathway In vivo and Increases Awake Phase Sleep
Authors: Subramaniyan Manivannan , Manivannan Sumithrra , Chelur Vikas , Tsetsenis Theodoros , Jiang Evan , Dani John A.
Published: May 2020

The hippocampus is essential for spatial learning and memory. To assess learning we used contextual fear conditioning (cFC), where animals learn to associate a place with aversive events like foot-shocks. Candidate memory mechanisms for cFC are long-term potentiation and long-term depression, but there is little direct evidence of them operating in the hippocampus in vivo following cFC. Also, little is known about the behavioral state changes induced by cFC. To address these issues, we recorded local field potentials in freely behaving mice by stimulating in the left dorsal CA1 region and recording in the right dorsal CA1 region. Synaptic strength in the commissural pathway was monitored by measuring field excitatory postsynaptic potentials (fEPSPs) before and after cFC. After cFC, the commissural pathway’s synaptic strength was potentiated. Although recordings occurred during the wake phase of the light/dark cycle, the mice slept more in the post-conditioning period than in the pre-conditioning period. Relative to awake periods, in non-rapid eye movement (NREM) sleep the fEPSPs were larger in both pre- and post-conditioning periods. We also found a significant negative correlation between the animal’s speed and fEPSP size. Therefore, to avoid confounds in the fEFSP potentiation estimates, we controlled for speed-related and sleep-related fEPSP changes and still found that cFC induced long-term potentiation, but no significant long-term depression. Synaptic strength changes were not found in the control group that simply explored the fear-conditioning chamber, indicating that exploration of the novel place did not produce the measurable effects caused by cFC. These results show that following cFC, the CA1 commissural pathway is potentiated, likely contributing to the functional integration of the left and right hippocampi in fear memory consolidation. In addition, the cFC paradigm produces significant changes in an animal’s behavioral state, which are observable as proximal changes in sleep patterns.
Satellite cell expansion is mediated by P-eIF2α dependentTacc3translation
Authors: Fujita Ryo , Lean Graham , Jamet Solène , Hébert Steven , Kleinman Claudia L. , Crist Colin
Published: May 2020

Translational control of gene expression is an important regulator of adult stem cell quiescence, activation and self-renewal. In skeletal muscle, quiescent satellite cells maintain low levels of protein synthesis, mediated in part through the phosphorylation of eIF2α (P-eIF2α). Pharmacological inhibition of the eIF2α phosphatase with the small molecule sal003 maintains P-eIF2α and permits the expansion of satellite cellsex vivo. Paradoxically, P-eIF2α also increases the translation of specific mRNAs, which is mediated by P-eIF2α dependent readthrough of inhibitory upstream open reading frames (uORFs). Here, we ask whether P-eIF2α dependent mRNA translation enables expansion of satellite cells. Using transcriptomic and proteomic analyses, we show a number of genes associated with the assembly of the spindle pole to be upregulated at the level of protein, without corresponding change in mRNA levels, in satellite cells expanded in the presence of sal003. We show that uORFs in the 5’UTR of mRNA for the mitotic spindle stability geneTacc3direct P-eIF2α dependent translation. Satellite cells deficient for TACC3 exhibit defects in expansion, self-renewal and regeneration of skeletal muscle.

Significance

Translational control of gene expression has emerged as an important regulator of adult stem cell populations, which maintain low levels of protein synthesis. In adult muscle stem cells, or satellite cells, a portrait of translational control has emerged whereby multiple repression mechanisms prevent the translation of specific mRNAs. It remains unclear how other mRNAs escape repression and are efficiently translated. We show that within the context of low global rates of protein synthesis, satellite cell expansion occurs through the selective translation ofTacc3mRNA.Tacc3deficient satellite cells expand poorly, leading to defects in skeletal muscle regeneration. Our study provides a more complete picture of translational control of gene expression in adult stem cell populations.
Methamphetamine enhances caveolar transport of therapeutic drugs across the rodent blood-brain barrier
Authors: Chang Jui-Hsien , Greene Chris , Futter Clare , Nichols Benjamin J. , Campbell Matthew , Turowski Patric
Published: May 2020

The blood-brain barrier (BBB) is a multifactorial and multicellular vascular interface separating the systemic environment from the central nervous system (CNS). It gates cerebral penetration of circulating molecules and cells and is the principal reason for low accumulation of many therapeutics in the brain. Low dose methamphetamine (METH) induces fluid phase transcytosis across the BBBin vitroand could therefore be used to enhance CNS drug delivery. Here we show, that low dose intravascular METH induced significant leakage exclusively via caveolar transport at the intact BBB in rodentsex vivo. Notably, METH-induced leakage was suppressed at 4°C and in Caveolin-1 (CAV1) knockout mice. Furthermore, METH strongly enhanced brain penetration of therapeutic molecules, namely doxorubicin (DOX), a small chemotherapeutic agent, and aflibercept (AFL), a ca. 100 kDA recombinant protein. Lastly, METH improved the therapeutic efficacy of DOX in a mouse model of human glioblastoma (GBM), as measured by a 25% increase in median survival time (p = 0.0024). Collectively, our data indicated that METH can facilitate preclinical assessment of novel experimental treatments and has the potential to enhance drug delivery to the diseased CNS.
Fast purification of recombinant monomeric amyloid-β fromE. coliand amyloid-β-mCherry aggregates from mammalian cells
Authors: Stephens Amberley D. , Lu Meng , Kaminski Schierle Gabriele S.
Published: May 2020

The Alzheimer’s disease related peptide, Amyloid-beta (Aβ)1-40 and 1-42, has proven difficult to be purified as a recombinant monomeric protein due its expression inE. colileading to the formation of insoluble inclusion bodies and its tendency to quickly form insoluble aggregates. A vast array of methods have been used so far, yet many have pitfalls, such as the use of tags for ease of Aβ isolation, the formation of Aβ multimers within the time frame of extraction or the need to reconstitute Aβ from a freeze dried state. Here, we present a rapid protocol to produce highly pure and monomeric recombinant Aβ using a one-step ion exchange purification method and to label the peptide using a maleimide dye. The solublisation and purification steps take only three hours. We also present a protocol for the isolation of Aβ-mCherry from mammalian cells.

Highlights

Purification of untagged, monomeric recombinant Aβ fromE. coli.

A fast protocol; 6 hours forE. coligrowth and Aβ expression, 2 hours to clean inclusion bodies, 45 mins to solublise and purify the peptide.

No freeze drying step that can lead to oligomer formation.

Purification of fluorescent Aβ-mCherry from mammalian cells.
MET Receptor Tyrosine Kinase Regulates Vagal Laryngeal Motor Neuron Development and Lifespan Ultrasonic Vocal Communication
Authors: Kamitakahara Anna K. , Marandi Ghoddousi Ramin Ali , Lanjewar Alexandra L. , Magalong Valerie M. , Wu Hsiao-Huei , Levitt Pat
Published: May 2020

The vagal motor nucleus ambiguus (nAmb) innervates the intrinsic muscles of the larynx, providing direct motor control over vocal production in humans and rodents. Here, we demonstrate that early developmental signaling through the MET receptor tyrosine kinase (MET) is required for proper formation of the nAmb. Embryonic deletion ofMetin the developing brainstem resulted in a loss of one-third of motor neurons in the nAmb. While the remaining neurons were able to establish connections with target muscles in the larynx, advanced signal processing analyses revealed severe deficits in ultrasonic vocalization in early postnatal life. Abnormal vocalization patterns persisted into adulthood in the majority of mice tested. Interestingly, 28% of adult mice recovered the ability to vocalize demonstrating heterogeneity in circuit restitution. Together, the data establish MET as a factor necessary for development of a specific subset of neurons in the nAmb required for normal ultrasonic vocalization.
PALLAS: Penalized mAximum LikeLihood and pArticle Swarms for Inference of Gene Regulatory Networks from Time Series Data
Authors: Tan Yukun , Neto Fernando B. Lima , Neto Ulisses Braga
Published: May 2020

We present PALLAS, a practical method for gene regulatory network (GRN) inference from time series data, which employs penalized maximum likelihood and particle swarms for optimization. PALLAS is based on the Partially-Observable Boolean Dynamical System (POBDS) model and thus does not require ad-hoc binarization of the data. The penalty in the likelihood is a LASSO regularization term, which encourages the resulting network to be sparse. PALLAS is able to scale to large networks under no prior knowledge, by virtue of a novel continuous-discrete Fish School Search particle swarm algorithm for efficient simultaneous maximization of the penalized likelihood over the discrete space of networks and the continuous space of observational parameters. The performance of PALLAS is demonstrated by a comprehensive set of experiments using synthetic data generated from real and artificial networks, as well as real time series microarray and RNA-seq data, where it is compared to several other well-known methods for gene regulatory network inference. The results show that PALLAS can infer GRNs efficiently and accurately. PALLAS is a fully-fledged program with a commandline user interface, written in python, and available on GitHub (https://github.com/yukuntan92/PALLAS).
ZEB1 is Required for NHEJ-Mediated DSB Repair in Euchromatin
Authors: Genetta Thomas L. , Abbas Tarek , Pandta Raj , Hunt Clayton R. , Pandita Tej. K. , Larner James M.
Published: May 2020

Ionizing radiation-induced DSBs are repaired primarily by the Non-Homologous End Joining (NHEJ) pathway, but the details of how this is regulated in different chromatin contexts are far from understood. We have discovered a novel response to DSBs that promotes NHEJ selectively in euchromatin, based on a novel interaction between the EMT-inducing transcriptional repressor ZEB1, and the well-studied NHEJ-promoting DNA repair factor 53BP1. Using a number of approaches, we have discovered that the ZEB1-53BP1 association is amplified following exposure of cells to IR and that they co-localize at IR-induced foci (IRIF). Depletion of ZEB1 enhances radio-sensitivity and increases IR-induced chromosomal aberrations in an ATM-independent manner. The very rapid recruitment–within 2 seconds–of ZEB1 to euchromatic DSBs is like-wise ATM-independent, but DNA-PK-dependent and is required for subsequent recruitment of 53BP1. ZEB1 promotes NHEJ and inhibits HR through its homeodomain by inducing 53BP1-permissive, pro-NHEJ/anti-HR chromatin modifications. Lastly, depletion of ZEB1 increases hyper-resection at DSBs and inhibits physiological DSB repair. These results support the argument that ZEB1 plays an essential role in DSB repair in euchromatin by establishing a 53BP1-permissive/pro-NHEJ chromatin environment.
Regional, layer, and cell-class specific connectivity of the mouse default mode network
Authors: Whitesell Jennifer D. , Liska Adam , Coletta Ludovico , Hirokawa Karla E. , Bohn Phillip , Williford Ali , Groblewski Peter A. , Graddis Nile , Kuan Leonard , Knox Joseph E. , Ho Anh , Wakeman Wayne , Nicovich Philip R. , Nguyen Thuc Nghi , Garren Emma , van Velthoven Cindy T. J. , Fong Olivia , Feng David , Naeemi Maitham , Henry Alex M. , Dee Nick , Smith Kimberly A. , Levi Boaz P. , Ng Lydia , Tasic Bosiljka , Zeng Hongkui , Mihalas Stefan , Gozzi Alessandro , Harris Julie A.
Published: May 2020

The evolutionarily conserved default mode network (DMN) is characterized by temporally correlated activity between brain regions during resting states. The DMN has emerged as a selectively vulnerable network in multiple disorders, so understanding its anatomical composition will provide fundamental insight into how its function is impacted by disease. Reproducible rodent analogs of the human DMN offer an opportunity to investigate the underlying brain regions and structural connectivity (SC) with high spatial and cell type resolution. Here, we performed systematic analyses using mouse resting state functional magnetic resonance imaging to identify the DMN and whole brain axonal tracing data, co-registered to the 3D Allen Mouse Common Coordinate Framework reference atlas. We identified the specific, predominantly cortical, brain regions comprising the mouse DMN and report preferential SC between these regions. Next, at the cell class level, we report that cortical layer (L) 2/3 neurons in DMN regions project almost exclusively to other DMN regions, whereas L5 neurons project to targets both in and out of the DMN. We then test the hypothesis that in- and out-DMN projection patterns originate from distinct L5 neuron sub-classes using an intersectional viral tracing strategy to label all the axons from neurons defined by a single target. In the ventral retrosplenial cortex, a core DMN region, we found two L5 projection types related to the DMN and mapped them to unique transcriptomically-defined cell types. Together, our results provide a multi-scale description of the anatomical correlates of the mouse DMN.
Reference-free Cell-type Annotation for Single-cell Transcriptomics using Deep Learning with a Weighted Graph Neural Network
Authors: Shao Xin , Yang Haihong , Zhuang Xiang , Liao Jie , Yang Yueren , Yang Penghui , Cheng Junyun , Lu Xiaoyan , Chen Huajun , Fan Xiaohui
Published: May 2020

Advances in single-cell RNA sequencing (scRNA-seq) have furthered the simultaneous classification of thousands of cells in a single assay based on transcriptome profiling. In most analysis protocols, single-cell type annotation relies on marker genes or RNA-seq profiles, resulting in poor extrapolation. Here, we introduce scDeepSort (https://github.com/ZJUFanLab/scDeepSort), a reference-free cell-type annotation tool for single-cell transcriptomics that uses a deep learning model with a weighted graph neural network. Using human and mouse scRNA-seq data resources, we demonstrate the feasibility of scDeepSort and its high accuracy in labeling 764,741 cells involving 56 human and 32 mouse tissues. Significantly, scDeepSort outperformed reference-dependent methods in annotating 76 external testing scRNA-seq datasets, including 126,384 cells (85.79%) from ten human tissues and 134,604 cells from 12 mouse tissues (81.30%). scDeepSort accurately revealed cell identities without prior reference knowledge, thus potentially providing new insights into mechanisms underlying biological processes, disease pathogenesis, and disease progression at a single-cell resolution.
Chromosome-level genome assembly of the African pike,Hepsetus odoe
Authors: Du Xiao , Hong Xiaoning , Fan Guangyi , Huang Xiaoyun , Sun Shuai , Bingjie Ouyang , Zhang He , Zhang Mengqi , Liu Shanshan , Liu Xin , Zhang Wenwei
Published: May 2020

The order Characiformes is one of the largest components of the freshwater teleost fauna inhabiting exclusively in South America and Africa with great ecological and economical significance. Yet, quite limited genomic resources are available to study this group and their transatlantic vicariance. In this study we present a chromosome-level genome assembly of the African pike (Hepsetus odoe), a representative member of the African Characiformes. To this end, we generated 119, 11, and 67 Gb reads using the single tube long fragment read (stLFR), Oxford Nanopore, and Hi-C sequencing technologies, respectively. We obtained an 862.1 Mb genome assembly with the contig and scaffold N50 of 347.4 kb and 25.8 Mb, respectively. Hi-C sequencing produced 29 chromosomes with 742.5 Mb, representing 86.1% of the genome. 24,314 protein-coding genes were predicted and 23,999 (98.7%) genes were functionally annotated. The chromosomal-scale genome assembly will be useful for functional and evolutionary studies of the African pike and promote the study of Characiformes speciation and evolution.
Nebulized in-line endotracheal dornase alfa and albuterol administered to mechanically ventilated COVID-19 patients: A case series
Authors: Weber Andrew G. , Chau Alice S , Egeblad Mikala , Barnes Betsy J , Janowitz Tobias
Published: May 2020

Background
Mechanically ventilated patients with coronavirus disease 2019 (COVID-19) have a mortality of 24-53%, in part due to distal mucopurulent secretions interfering with ventilation. Dornase alfa is recombinant human DNase 1 and digests DNA in mucoid sputum. Nebulized dornase alfa is FDA-approved for cystic fibrosis treatment. DNA from neutrophil extracellular traps (NETs) contributes to the viscosity of mucopurulent secretions. NETs are found in the serum of patients with severe COVID-19, and targeting NETs reduces mortality in animal models of acute respiratory distress syndrome (ARDS). Thus, dornase alfa may be beneficial to patients with severe COVID-19, acting as a mucolytic and targeting NETs. However, delivery of nebulized drugs can aerosolize SARS-CoV-2, which causes COVID-19, increasing the infection risk for staff. Here, we report a single center case series where dornase alfa was administered through an in-line nebulizer system to minimize risk of virus aerosolization.

Methods
Demographic, clinical data, and outcomes were collected from the electronic medical records of five mechanically ventilated patients with COVID-19, including three requiring veno-venous extracorporeal membrane oxygenation (VV-ECMO), treated with nebulized in-line endotracheal dornase alfa co-administered with albuterol (used to increase delivery to the alveoli), between March 31 and April 24, 2020. Data on tolerability and responses, including longitudinal values capturing respiratory function and inflammatory status, were analyzed.

Results
Following nebulized in-line administration of dornase alfa with albuterol, the fraction of inspired oxygen requirements was reduced for all five patients. All patients remain alive and two patients have been discharged from the intensive care unit. No drug associated toxicities were identified.

Conclusions
The results presented in this case series suggest that dornase alfa will be well-tolerated by critically ill patients with COVID-19. Clinical trials are required to formally test the dosing, safety, and efficacy of dornase alfa in COVID-19, and two have recently been registered (NCT04359654 and NCT04355364). With this case series, we hope to contribute to the development of management approaches for critically ill patients with COVID-19.
Evolutionary Dynamics And Geographic Dispersal Of Beta Coronaviruses In African Bats
Authors: Motayo Babatunde Olanrewaju , Oluwasemowo Olukunle Oluwapamilerin , Akinduti Paul Akiniyi
Published: May 2020

Bats have been shown to serve as reservoir host of various viral agents including coronaviruses. They have also been associated with the novel coronavirus SARS-CoV-2. This has made them an all important agent for CoV evolution and transmission. Our objective in this study was to investigate the dispersal, phylogenomics and evolution ofbetacoronavirus(βCoV) among African bats. We retrieved sequence data from established databases such as GenBank and Virus Pathogen Resource, covering the partial RNA dependent RNA polymerase (RdRP) gene of Bat coronaviruses from eight African, three Asian, five European, two South American countries and Australia. We analyzed for Phylogeographic information relating to genetic diversity and evolutionary dynamics. Our study revealed that majority of the African strains fell withinNorbecovirussubgenera, with an Evolutionary rate of 1.301 × 10−3, HPD (1.064 × 10−3– 1.434 × 10−3) subs/site/year. The African strains diversified into three main subgenera,Norbecovirus, HibecovirusandMarbecovirus. The time to most common recent ancestor forNorbecovirusstrains was 1968, and 2010, for the AfricanMarbecovirusstrains. There was evidence of inter species transmission ofNorbecovirusamong bats in Cameroun and DRC. Phlylogeography showed that there were inter-continental spread of Bt-CoV from Europe, China and Hong Kong into Central and Southern Africa, highlighting the possibility of long distance transmission. Our study has elucidated the possible evolutionary origins of βCoV among African bats, we therefore advocate for broader studies of whole genome sequences of BtCoV to further understand the drivers for their emergence and zoonotic spillovers into human population.
Meanders as a scaling motif for understanding of floodplain soil microbiome and biogeochemical potential at the watershed scale
Authors: Carnevali Paula B. Matheus , Lavy Adi , Thomas Alex D. , Crits-Christoph Alexander , Diamond Spencer , Meéheust Raphaeël , Olm Matthew R. , Sharrar Allison , Lei Shufei , Dong Wenming , Falco Nicola , Bouskill Nicholas , Newcomer Michelle , Nico Peter , Wainwright Haruko , Dwivedi Dipankar , Williams Kenneth H. , Hubbard Susan , Banfield Jillian F.
Published: May 2020

Biogeochemical exports of C, N, S and H2from watersheds are modulated by the activity of microorganisms that function over micron scales. This disparity of scales presents a substantial challenge for development of predictive models describing watershed function. Here, we tested the hypothesis that meander-bound regions exhibit patterns of microbial metabolic potential that are broadly predictive of biogeochemical processes in floodplain soils along a river corridor. We intensively sampled floodplain soils located in the upper, middle, and lower reaches of the East River in Colorado and reconstructed 248 draft quality genomes representative at a sub-species level. Approximately one third of the representative genomes were detected across all three locations with similar levels of abundance, and despite the very high microbial diversity and complexity of the soils, ~15% of species were detected in two consecutive years. A core floodplain microbiome was enriched in bacterial capacities for aerobic respiration, aerobic CO oxidation, and thiosulfate oxidation with the formation of elemental sulfur. We did not detect systematic patterns of gene abundance based on sampling position relative to the river. However, at the watershed scale meander-bound floodplains appear to serve as scaling motifs that predict aggregate capacities for biogeochemical transformations in floodplain soils. Given this, we conducted a transcriptomic analysis of the middle site. Overall, the most highly transcribed genes wereamoCABandnxrAB(for nitrification) followed by genes involved in methanol and formate oxidation, and nitrogen and CO2fixation. Low soil organic carbon correlated with high activity of genes involved in methanol, formate, sulfide, hydrogen, and ammonia oxidation, nitrite oxidoreduction, and nitrate and nitrite reduction. Thus, widely represented genetic capacities did not predictin situactivity at one time point, but rather they define a reservoir of biogeochemical potential available as conditions change.
Visualizing cassava bacterial blight at the molecular level using CRISPR-mediated homology-directed repair
Authors: Veley Kira M. , Okwuonu Ihuoma , Jensen Greg , Yoder Marisa , Taylor Nigel J. , Meyers Blake C. , Bart Rebecca S.
Published: May 2020

Research on a few model, plant-pathogen systems has benefitted from years of tool and resource development. This is not the case for the vast majority of economically and nutritionally important plants, creating a crop improvement bottleneck. Cassava bacterial blight (CBB), caused byXanthomonas axonopodispv.manihotis(Xam), is an important disease in all regions where cassava (Manihot esculentaCrantz) is grown. Here we describe development of a tool for molecular-level visualization of CBB dynamicsin vivo. Using CRISPR-mediated homology-directed repair (HDR), we generated plants containing scarless insertion of a GFP reporter at the CBB susceptibility (S) geneMeSWEET10a. Activation ofMeSWEET10a-GFPbyXamwas subsequently visualized at transcriptional and translational levels. Development of this tool was facilitated by a time-saving, adaptable strategy for identifying successful products of HDR, currently a limiting factor in plant research. This strategy has the potential to enable such research in other systems, improving the practicality of HDR-based experimentation.
ACE2-Variants Indicate Potential SARS-CoV-2-Susceptibility in Animals: An Extensive Molecular Dynamics Study
Authors: Pach Szymon , Nguyen Trung Ngoc , Trimpert Jakob , Kunec Dusan , Osterrieder Nikolaus , Wolber Gerhard
Published: May 2020

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in late 2019 and since evolved into a global threat with nearly 4.4 million infected people and over 290,000 confirmed deaths worldwide.1 SARS-CoV-2 is an enveloped virus presenting spike (S) glycoproteins on its outer surface. Binding of S to host cell angiotensin converting enzyme 2 (ACE2) is thought to be critical for cellular entry. The host range of the virus extends far beyond humans and non-human primates. Natural and experimental infections have confirmed high susceptibility of cats, ferrets, and hamsters, whereas dogs, mice, rats, pigs, and chickens seem refractory to SARS-CoV-2 infection. To investigate the reason for the variable susceptibility observed in different species, we have developed molecular descriptors to efficiently analyze our dynamic simulation models of complexes between SARS-CoV-2 S and ACE2. Based on our analyses we predict that: (i) the red squirrel is likely susceptible to SARS-CoV-2 infection and (ii) specific mutations in ACE2 of dogs, rats, and mice render them susceptible to SARS-CoV-2 infection.
An NMR-based biosensor to measure stereo-specific methionine sulfoxide reductase (MSR) activitiesin vitroandin vivo
Authors: Sánchez-López Carolina , Labadie Natalia , Lombardo Verónica A. , Biglione Franco A. , Manta Bruno , Jacob Reeba S. , Gladyshev Vadim N. , Abdelilah-Seyfried Salim , Selenko Philipp , Binolfi Andres
Published: May 2020

Oxidation of protein methionines to methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to methionine by two families of conserved methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target theS- orR-diastereoisomers of methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments that range from native cell lysates to zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways. Our approach further extends high-resolution in-cell NMR measurements of exogenously delivered biomolecules to an entire multicellular organism.
The cIAP ubiquitin ligases sustain type 3 γδ T and innate lymphoid cells during aging to allow normal cutaneous and mucosal responses
Authors: Rizk John , Mörbe Urs M. , Agerholm Rasmus , Ulmert Isabel , Tardos Elisa Catafal , Kadekar Darshana , Baglioni Maria V. , Viñals Monica Torrellas , Bekiaris Vasileios
Published: May 2020

Environmental and molecular cues early in life are often associated with the permanent shaping of our immune system during adulthood. Although increasing, our knowledge of the signaling pathways that operate in early life and their temporal mode of action is limited. Herein, we demonstrate that the cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2), which are E3 ubiquitin ligases and master regulators of the nuclear factor-kappa B (NF-κB) pathway, function during late neonatal and prepubescent life to sustain interleukin(IL)-17-producing gamma delta T cells (γδT17) and group 3 innate lymphoid cells (ILC3). We show that cell-intrinsic deficiency in cIAP1/2 at 3-4 weeks of life leads to downregulation of the transcription factors cMAF and RORγt, and failure to enter cytokine-induced cell cycle. This is followed by progressive loss of γδT17 cells and ILC3 while mice are aging. Mice deficient in cIAP1/2 have severely reduced γδT17 cells and ILC3, present with suboptimal γδT17 responses in the skin, lack small intestinal isolated lymphoid follicles and cannot control intestinal bacterial infection. Mechanistically, these effects appear to be dependent on overt activation of the non-canonical NF-κB pathway. Our data identify the cIAP E3 ubiquitin ligases as critical early life molecular switches for establishing effective type-3 immunity during aging.
Identification of conserved epitopes in SARS-CoV-2 spike and nucleocapsid protein
Authors: Forcelloni Sergio , Benedetti Anna , Dilucca Maddalena , Giansanti Andrea
Published: May 2020

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which first occurred in Wuhan (China) in December 2019, is a novel virus that causes a severe acute respiratory disease. The virus spike glycoproteins and nucleocapsid proteins are the main targets for the development of vaccines and antiviral drugs, to control the disease spread. We herein study the structural order-disorder propensity and the rates of evolution of these two proteins to characterize their B cell and T cell epitopes, previously suggested to contribute to immune response caused by SARS-CoV-2 infections. We first analyzed the rates of evolution along the sequences of spike and nucleocapsid proteins in relation to the spatial locations of their epitopes. For this purpose, we compared orthologs from seven human coronaviruses: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. We then focus on the local, structural order-disorder propensities of the protein regions where the SARS-CoV-2 epitopes are located. We show that the vast majority of nucleocapsid protein epitopes overlap the RNA-binding and dimerization domains and some of them are characterized by low rates of evolutions. Similarly, spike protein epitopes are preferentially located in regions that are predicted to be ordered and well-conserved, in correspondence of the heptad repeats 1 and 2. Interestingly, both the receptor-binding motif to ACE2 and the fusion peptide of spike protein are characterized by high rates of evolution, probably to overcome host immunity. In conclusion, our results provide evidence for conserved epitopes that may help to develop long-lasting, broad-spectrum SARS-CoV-2 vaccines.
Identification ofAvramr1fromPhytophthora infestansusing long read and cDNA pathogen-enrichment sequencing (PenSeq)
Authors: Lin Xiao , Song Tianqiao , Fairhead Sebastian , Witek Kamil , Jouet Agathe , Jupe Florian , Witek Agnieszka I. , Karki Hari S. , Vleeshouwers Vivianne G. A. A. , Hein Ingo , Jones Jonathan D. G.
Published: May 2020

Potato late blight, caused by the oomycete pathogenPhytophthora infestans, significantly hampers potato production. Recently, a newResistance to Phytophthora infestans(Rpi) gene,Rpi-amr1, was cloned from a wildSolanumspecies,Solanum americanum. Identification of the corresponding recognized effector (Avirulence, orAvr)genes fromP. infestansis key to elucidating their naturally occurring sequence variation, which in turn informs the potential durability of the cognate late blight resistance.

To identify theP. infestanseffector recognized byRpi-amr1, we screened available effector libraries and used long read and cDNA pathogen-enrichment sequencing (PenSeq) on fourP. infestansisolates to explore the untested effectors.

By using SMRT and cDNA PenSeq, we identified 47 highly expressed effectors fromP. infestans, including PITG_07569 which triggers a highly specific cell death response when transiently co-expressed withRpi-amr1inNicotiana benthamiana, suggesting thatPITG_07569isAvramr1.

Here we demonstrate that long read and cDNA PenSeq enables the identification of full-length RxLR effector families, and their expression profile. This study has revealed key insights into the evolution and polymorphism of a complex RxLR effector family that is associated with the recognition byRpi-amr1.
Intraflagellar transport during the assembly of flagella of different length inTrypanosoma bruceiisolated from tsetse flies
Authors: Bertiaux Eloïse , Mallet Adeline , Rotureau Brice , Bastin Philippe
Published: May 2020

Multicellular organisms assemble cilia and flagella of precise lengths differing from one cell to another, yet little is known about the mechanisms governing these differences. Similarly, protists assemble flagella of different lengths according to the stage of their life cycle. This is the case ofTrypanosoma bruceithat assembles flagella of 3 to 30 µm during its development in the tsetse fly. It provides an opportunity to examine how cells naturally modulate organelle length. Flagella are constructed by addition of new blocks at their distal end via intraflagellar transport (IFT). Immunofluorescence assays, 3-D electron microscopy and live cell imaging revealed that IFT was present in all life cycle stages. IFT proteins are concentrated at the base, IFT trains are located along doublets 3-4 & 7-8 and travel bidirectionally in the flagellum. Quantitative analysis demonstrated that the total amount of IFT proteins correlates with the length of the flagellum. Surprisingly, the shortest flagellum exhibited a supplementary large amount of dynamic IFT material at its distal end. The contribution of IFT and other factors to the regulation of flagellum length is discussed.

Summary statement

This work investigated the assembly of flagella of different length during the development ofTrypanosoma bruceiin the tsetse fly, revealing a direct correlation between the amount of intraflagellar transport proteins and flagellum length.
A role for glutathione in buffering excess intracellular copper inStreptococcus pyogenes
Authors: Stewart Louisa J. , Ong Cheryl-lynn Y. , Zhang May M. , Brouwer Stephan , McIntyre Liam , Davies Mark R. , Walker Mark J. , McEwan Alastair G. , Waldron Kevin J. , Djoko Karrera Y.
Published: May 2020

Copper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally-responsive system for Cu export. In the Gram-positive human pathogenStreptococcus pyogenes(Group AStreptococcus, GAS), this system is encoded by thecopYAZoperon. In this study, we demonstrate that the site of GAS infectionin vivorepresents a Cu-rich environment but inactivation of thecopACu efflux gene does not reduce virulence in a mouse model of invasive disease.In vitro, Cu treatment leads to multiple observable phenotypes, including defects in growth and viability, decreased fermentation, inhibition of glyceraldehyde 3-phosphate dehydrogenase (GapA) activity, and misregulation of metal homeostasis, likely as a consequence of mismetalation of non-cognate metal-binding sites. Surprisingly, the onset of these effects is delayed by ∼4 h even though expression ofcopZis upregulated immediately upon exposure to Cu. We further show that the onset of all phenotypes coincides with depletion of intracellular glutathione (GSH). Supplementation with extracellular GSH replenishes the intracellular pool of this thiol and suppresses all the observable effects of Cu treatment. Our results indicate that GSH contributes to buffering of excess intracellular Cu when the transcriptionally-responsive Cu export system is overwhelmed. Thus, while thecopYAZoperon is responsible for Cuhomeostasis, GSH has a role in Cutolerancethat allows bacteria to maintain metabolism even in the presence of an excess of this metal ion. This study advances fundamental understanding of Cu handling in the bacterial cytoplasm.

IMPORTANCE

The control of intracellular metal availability is fundamental to bacterial physiology. In the case of copper (Cu), it is established that rising intracellular Cu levels eventually fill the metal-sensing site of the endogenous Cu-sensing transcriptional regulator, which in turn induces transcription of a copper export pump. This response caps intracellular Cu availability below a well-defined threshold and prevents Cu toxicity. Glutathione, abundant in many bacteria, is known to bind Cu and is long assumed to contribute to bacterial Cu handling. However, there is some ambiguity since neither its biosynthesis nor uptake is Cu-regulated. Furthermore, there is little experimental support for this role of glutathione beyond measurement of the effect of Cu on growth of glutathione-deficient mutants. Our work with Group AStreptococcusprovides new evidence that glutathione increases the threshold of intracellular Cu availability that can be tolerated by bacteria and thus advances fundamental understanding of bacterial Cu handling.
GATA-1-dependent histone H3K27ac mediates erythroid cell-specific interaction between CTCF sites
Authors: Kim Yea Woon , Kang Yujin , Kang Jin , Kim AeRi
Published: May 2020

CTCF sites interact with each other in the chromatin environment, establishing domains. Our previous study showed that interaction between CTCF sites is cell type-specific around the β-globin locus and is dependent on erythroid specific activator GATA-1. To find out molecular mechanisms of the cell type-specific interaction, we directly inhibited GATA-1 binding to the β-globin enhancers by deleting its binding motifs and found that histone H3K27 acetylation (H3K27ac) was decreased at CTCF sites surrounding the β-globin locus, even though CTCF binding itself was maintained at the sites. Forced H3K27ac by TSA treatment or CBP/p300 KD affected the interactions between CTCF sites around the β-globin locus. Analysis of public ChIA-PET data revealed that H3K27ac is higher at CTCF sites forming short chromatin interactions than long interactions. The short interactions contain erythropoiesis-associated genes and GATA-1 binding sites in erythroid K562 cells. Depletion of GATA-1 reduced H3K27ac at CTCF sites near erythroid enhancers. These results indicate that GATA-1-dependent histone H3K27ac at neighboring CTCF sites mediates erythroid specific chromatin interaction between them.
Validation of suitable genes for normalization of diurnal gene expression studies inChenopodium quinoa
Authors: Maldonado-Taipe Nathaly , Sarange Dilan , Schmöckel Sandra , Jung Christian , Emrani Nazgol
Published: May 2020

Quinoa depicts high nutritional quality and abiotic stress resistance attracting strong interest in the last years. To unravel the function of candidate genes for agronomically relevant traits, studying their transcriptional activities by RT-qPCR is an important experimental approach. The accuracy of such experiments strongly depends on precise data normalization. To date, validation of potential candidate genes for normalization of diurnal expression studies has not been performed inC. quinoa. We selected eight candidate genes based on transcriptome data and literature survey, including conventionally used reference genes. We used three statistical algorithms (BestKeeper, geNorm and NormFinder) to test their stability and added further validation by a simulation-based strategy. We demonstrated that using different reference genes, including those top ranked by stability, causes significant differences among the resulting diurnal expression patterns, and that our novel approach overcomes failures related to stability-based selection of reference genes. Our results show that isocitrate dehydrogenase enzyme (IDH-A) and polypyrimidine tract-binding protein (PTB) are suitable genes to normalize diurnal expression data of two different quinoa accessions. The validated reference genes obtained in this study will improve the accuracy of RT-qPCR data normalization and facilitate gene expression studies in quinoa.
Plasticity of the xylem vulnerability to embolism in poplar relies on quantitative pit properties rather than on pit structure
Authors: Lemaire Cédric , Quilichini Yann , Brunel-Michac Nicole , Santini Jérémie , Berti Liliane , Cartailler Julien , Conchon Pierre , Badel Éric , Herbette Stéphane
Published: May 2020

Knowledge on variations of drought resistance traits are needed to predict the potential of trees to acclimate to coming severe drought events. Xylem vulnerability to embolism is a key parameter related to such droughts, and its phenotypic variability relies mainly on environmental plasticity. We investigated the structural determinants controlling the plasticity of vulnerability to embolism, focusing on the key elements involved in the air bubble entry in a vessel, especially the inter-vessel pits. Poplar saplings (Populus tremula x alba) grown in contrasted water availability or light exposure exhibited differences in vulnerability to embolism in a range of 0.76 MPa. We then characterized the structural changes related to qualitative and quantitative pit characteristics, from the pit structure to the organization of xylem vessels, using different microscopy techniques (TEM, SEM, light). X-ray microtomography analysis allowed observing the vessel vulnerability and testing some of the relationships between structural traits and vulnerability to embolism inside the xylem. The pit ultrastructure did not change, whereas the vessel dimensions increased with vulnerability to embolism and the grouping index and fraction of inter-vessel cell wall decreased with vulnerability to embolism. These findings holds when comparing trees or when comparing vessels inside the xylem. These results evidenced that plasticity of vulnerability to embolism occurs through changes in the quantitative pit properties such as pit area and vessel grouping rather than on the pit structure.
Functional Exploration of Heterotrimeric Kinesin-II in IFT and Ciliary Length Control inChlamydomonas
Authors: Li Shufen , Chen Wei , Wan Kirsty Y , Tao Hui , Liang Xin , Pan Junmin
Published: May 2020

Heterotrimeric organization of kinesin-II is essential for its function in anterograde IFT in ciliogenesis. However, the molecular basis of forming this complex for its function is not well understood. In addition, the anterograde IFT velocity varies significantly in different organisms, but how motor speed affects ciliary length is not clear. We show thatChlamydomonaskinesin-II (CrKinesin-II) involves distinct mechanisms from mammals and C.elegansin its assembly to necessitate its function in IFT. Furthermore, chimeric CrKinesin-II with human kinesin-II motor domains functionedin vitroandin vivo, leading to a ~2.8-fold reduced anterograde IFT velocity and a similar fold reduction in IFT injection rate that supposedly correlates with ciliary assembly activity. However, the ciliary length was only mildly reduced (~15%). Modelling analyses suggest that such a non-linear scaling relationship between IFT velocity and ciliary length can be accounted for by limitation of the motors and/or its ciliary cargoes, e.g. tubulin.
Characteristic and quantifiable COVID-19-like abnormalities in CT- and PET/CT-imaged lungs of SARS-CoV-2-infected crab-eating macaques (Macaca fascicularis)
Authors: Finch Courtney L. , Crozier Ian , Lee Ji Hyun , Byrum Russ , Cooper Timothy K. , Liang Janie , Sharer Kaleb , Solomon Jeffrey , Sayre Philip J. , Kocher Gregory , Bartos Christopher , Aiosa Nina M. , Castro Marcelo , Larson Peter A. , Adams Ricky , Beitzel Brett , Di Paola Nicholas , Kugelman Jeffrey R. , Kurtz Jonathan R. , Burdette Tracey , Nason Martha C. , Feuerstein Irwin M. , Palacios Gustavo , Claire Marisa C. St. , Lackemeyer Matthew G. , Johnson Reed F. , Braun Katarina M. , Ramuta Mitchell D. , Wada Jiro , Schmaljohn Connie S. , Friedrich Thomas C. , O’Connor David H. , Kuhn Jens H.
Published: May 2020

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing an exponentially increasing number of coronavirus disease 19 (COVID-19) cases globally. Prioritization of medical countermeasures for evaluation in randomized clinical trials is critically hindered by the lack of COVID-19 animal models that enable accurate, quantifiable, and reproducible measurement of COVID-19 pulmonary disease free from observer bias. We first used serial computed tomography (CT) to demonstrate that bilateral intrabronchial instillation of SARS-CoV-2 into crab-eating macaques (Macaca fascicularis) results in mild-to-moderate lung abnormalities qualitatively characteristic of subclinical or mild-to-moderate COVID-19 (e.g., ground-glass opacities with or without reticulation, paving, or alveolar consolidation, peri-bronchial thickening, linear opacities) at typical locations (peripheral>central, posterior and dependent, bilateral, multi-lobar). We then used positron emission tomography (PET) analysis to demonstrate increased FDG uptake in the CT-defined lung abnormalities and regional lymph nodes. PET/CT imaging findings appeared in all macaques as early as 2 days post-exposure, variably progressed, and subsequently resolved by 6–12 days post-exposure. Finally, we applied operator-independent, semi-automatic quantification of the volume and radiodensity of CT abnormalities as a possible primary endpoint for immediate and objective efficacy testing of candidate medical countermeasures.