bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of downstream effectors of retinoic acid specifying the zebrafish pancreas by integrative genomics
Authors: López-Pérez Ana R. , Balwierz Piotr J. , Lenhard Boris , Muller Ferenc , Wardle Fiona C. , Manfroid Isabelle , Voz Marianne L. , Peers Bernard
Published: November 2020

Retinoic acid (RA) is a key signal for the specification of the pancreas. Still, the gene regulatory cascade triggered by RA in the endoderm remains poorly characterized. In this study, we investigated this regulatory network in zebrafish by combining RNA-seq, RAR ChIP-seq and ATAC-seq assays. By analysing the effect of RA and of the RA receptor (RAR) antagonist BMS439 on the transcriptome and on the chromatin accessibility of endodermal cells, we identified a large set of genes and regulatory regions regulated by RA signalling. RAR ChIP-seq further defined the direct RAR target genes including the knownhoxgenes as well as several pancreatic regulators likemnx1, insm1b, hnf1baandgata6. Comparison of our zebrafish data with available murine RAR ChIP-seq data highlighted conserved direct target genes and revealed that some RAR sites are under strong evolutionary constraints. Among them, a novel highly conserved RAR-induced enhancer was identified downstream of theHoxBlocus and driving expression in the nervous system and in the gut in a RA-dependant manner. Finally, ATAC-seq data unveiled the role of the RAR-direct targets Hnf1ba and Gata6 in opening chromatin at many regulatory loci upon RA treatment.

Summary statement

Combination of RNA-seq, ChIP-seq and ATAC-seq assays identifies genes directly and indirectly regulated by RA signalling in zebrafish endoderm. Comparison with murine data highlights RAR binding sites conserved among vertebrates.
The self-peptide repertoire plays a critical role in transplant tolerance induction
Authors: Son Eric T. , Faridi Pouya , Paul-Heng Moumita , Leong Mario , English Kieran , Ramarathinam Sri H. , Braun Asolina , Dudek Nadine L. , Alexander Ian E. , Lisowski Leszek , Bertolino Patrick , Bowen David G. , Purcell Anthony W. , Mifsud Nicole A. , Sharland Alexandra F.
Published: November 2020

While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly-alloreactive CD8+T cells have not been defined. In this study, we used a combination of genetically-engineered MHC class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly-recognised pMHC epitopes, and identified 17 strongly immunogenic H-2Kb-associated peptides recognised by CD8+T cells from B10.BR (H-2k) mice, 13 of which were also recognised by BALB/c (H-2d) mice. As few as five different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse.
Chromosome-levelde novoassembly ofCoprinopsis cinerea A43mut B43mut pab1-1#326 and genetic variant identification of mutants using Nanopore MinION sequencing
Authors: Xie Yichun , Zhong Yiyi , Chang Jinhui , Kwan Hoi Shan
Published: November 2020

The homokaryoticCoprinopsis cinereastrainA43mut B43mut pab1-1#326 is a widely used experimental model for developmental studies in mushroom-forming fungi. It can grow on defined artificial media and complete the whole lifecycle within two weeks. The mutations in mating type factorsAandBresult in the special feature of clamp formation and fruiting without mating. This feature allows investigations and manipulations with a homokaryotic genetic background. Current genome assembly of strain #326 was based on short-read sequencing data and was highly fragmented, leading to the bias in gene annotation and downstream analyses. Here, we report a chromosome-level genome assembly of strain #326. Oxford Nanopore Technology (ONT) MinION sequencing was used to get long reads. Illumina short reads was used to polish the sequences. A combined assembly yield 13 chromosomes and a mitochondrial genome as individual scaffolds. The assembly has 15,250 annotated genes with a high synteny with theC. cinereastrain Okayama-7 #130. This assembly has great improvement on contiguity and annotations. It is a suitable reference for further genomic studies, especially for the genetic, genomic and transcriptomic analyses in ONT long reads. Single nucleotide variants and structural variants in six mutagenized and cisplatin-screened mutants could be identified and validated. A 66 bp deletion in Ras GTPase-activating protein(RasGAP)was found in all mutants. To make a better use of ONT sequencing platform, we modified a high-molecular-weight genomic DNA isolation protocol based on magnetic beads for filamentous fungi. This study showed the use of MinION to construct a fungal reference genome and to perform downstream studies in an individual laboratory. An experimental workflow was proposed, from DNA isolation and whole genome sequencing, to genome assembly and variant calling. Our results provided solutions and parameters for fungal genomic analysis on MinION sequencing platform.

Highlight

A chromosome-level genome assembly ofC. cinerea #326

A fast and efficient high-molecular-weight fungal genomic DNA isolation protocol

Structural variant and single nucleotide variant calling using Nanopore reads

A series of solutions and reference parameters for fungal genomic analysis on MinION
Characterisation of bacteriophage JD419, a Staphylococcal phage with an unusual morphology and broad host range
Authors: Feng Tingting , Leptihn Sebastian , Dong Ke , Loh Belinda , Zhang Yan , Li Mingyue , Guo Xiaokui , Cui Zelin
Published: November 2020

As an antimicrobial therapy, therapeutic phages, also known as “Phage therapy” are able to inactivate multi-drug resistant bacteria such as methicillin and vancomycin resistantS. aureusand thus present a possible treatment for infections that are otherwise incurable. In this paper, we present a novel phage called JD419, which has a remarkably wide host-range. The virulent phage JD419 exhibits an elongated capsid and was able to infect and lyse 83 of all 129 tested clinical strains (64.3%) of multi-drug resistantS. aureusincluding MRSA. To evaluate the potential as a therapeutic phage, we tested the ability of phage JD419 to remain infectious after treatment exceeding physiological pH or temperature. The lytic activity of the phage was retained at pH values of 6.0-8.0 and at temperatures below 50°C. As phages sometimes contain virulence genes, we sequenced the complete genome of JD419. The 45509 bp genome contains a predicted 65 ORFs, none of which show homology to any known virulence or antibiotic resistance genes. Our study illustrates thatStaphylococcusphage JD419 has the potential to be used for diagnostic, prophylaxic and therapeutic purposes.
Nafamostat Mesylate in lipid carrier for nasal SARS-CoV2 titer reduction in a hamster model
Authors: Cornelissen Lisette , Hoefsmit Esmee , Rao Disha , Lijnsvelt Judith , van Keulen Lucien , van Es Marieke , Grimm Volker , Medema René H. , Blank Christian U.
Published: November 2020

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) has been responsible for the largest pandemic in recent decades. After seemingly being in control due to consequent lock-downs and social distancing, the majority of countries faces currently a second wave of exponentially increasing infections, hospital referrals and deaths due to SARS-CoV-2-mediated disease (COVID-19). To date, no effective vaccination has been found, and wearing masks and social distancing are the only effective approaches to reduce further spreading.

However, unwillingness in the societies to distance again and consequently wear masks might be reasons for the second SARS-CoV-2 infection wave. User-friendly chemicals interfering at the host site with viral entry might be an approach to contain the pandemic. In addition, such an approach would work synergistic with vaccinations that miss new virus mutants.

Nafamostat (NM) has been shownin vitroto interfere with cellular virus entry by inhibition of the host transmembrane protease serine 2 (TMPRSS2), an enzyme required for SARS-CoV-2 spike protein cleavage, a prerequisite for cell entry.

We hypothesized that nasal application of NM in a liposomal layer (as additional mechanical barrier) could lower the nasal viral load and subsequently reduce the severity of COVID-19. We found, indeed, that nasal viral load one day post single NM application, was lowered in a hamster SARS-CoV-2 infection model. However, severity of subsequent local tissue destruction and weight loss due to pneumonitis was not favorably altered.

In conclusion, a single NM application reduced nasal viral load, but did not favorably improve the outcome of COVID-19, likely due to the short half-time of NM. Improvement of NM stability or repetitive application (which was not permitted in this animal model according to Dutch law) might circumvent these challenges.
Novel drivers of virulence inClostridioides difficileidentified via context-specific metabolic network analysis
Authors: Jenior Matthew L , Leslie Jhansi L , Powers Deborah A , Garrett Elizabeth M , Walker Kimberly A , Dickenson Mary E , Petri William A , Tamayo Rita , Papin Jason A
Published: November 2020

The pathogenClostridioides difficilecauses toxin-mediated diarrhea and is the leading cause of hospital-acquired infection in the US. Due to growing antibiotic resistance and recurrent infection, targetingC. difficilemetabolism presents a new approach to combat this infection. Genome-scale metabolic network reconstructions (GENREs) have been used to identify therapeutic targets and uncover properties that determine cellular behaviors. Thus, we constructedC. difficileGENREs for a hyper-virulent isolate (str. R20291) and a historic strain (str. 630), validating both within vitroandin vivodatasets. Growth simulations revealed significant correlations with measured carbon source usage (PPV ≥ 92.7%), and single-gene deletion analysis showed >89.0% accuracy. Next, we utilized each GENRE to identify metabolic drivers of both sporulation and biofilm formation. Through contextualization of each model using transcriptomes generated fromin vitroand infection conditions, we discovered reliance on the Pentose Phosphate Pathway as well as increased usage of cytidine and N-acetylneuraminate when virulence expression is reduced, which was subsequently supported experimentally. Our results highlight the ability of GENREs to identify novel metabolite signals in bacterial pathogenesis.

Importance

Clostridioides difficileis a Gram-positive, sporulating anaerobe that has become the leading cause of hospital-acquired infections. Numerous studies have demonstrated the importance of specific metabolic pathways in aspects ofC. difficilepathophysiology, from initial colonization to regulation of virulence factors. In the past, genome-scale metabolic network reconstruction (GENRE) analysis of bacteria has enabled systematic investigation of the genetic and metabolic properties that contribute to downstream virulence phenotypes. With this in mind, we generated and extensively curatedC. difficileGENREs for both a well-studied laboratory strain (str. 630) as well as a more recently characterized hyper-virulent isolate (str. R20291).In silicovalidation of both GENREs revealed high degrees of agreement with experimental gene essentiality and carbon source utilization datasets. Subsequent exploration of context-specific metabolism during bothin vitrogrowth and infection revealed consistent patterns of metabolism which corresponded with experimentally measured increases in virulence factor expression. Our results support that differentialC. difficilevirulence is associated with distinct metabolic programs related use of carbon sources and provides a platform for identification of novel therapeutic targets.
Inherent protection of bacteria from beta-lactam antibiotics by wet-dry cycles with microscopic surface wetness
Authors: Beizman-Magen Yana , Grinberg Maor , Orevi Tomer , Kashtan Nadav
Published: November 2020

A large portion of bacterial life occurs on surfaces that are not constantly saturated with water and experience recurrent wet-dry cycles. While soil, plant leaves and roots, and many indoor surfaces may appear dry when not saturated with water, they are in fact often covered by thin liquid films and microdroplets, invisible to the naked eye, known as microscopic surface wetness (MSW). Such MSW, resulting from the condensation of water vapor to hygroscopic salts, is ubiquitous yet largely underexplored. A wide variety of antibiotics are abundant in environments where MSW occurs, yet little is known about bacterial response to antibiotics in wet-dry cycles and under MSW conditions. UsingE. colias a model organism, we show, through a combination of experiments and computational modeling, that bacteria are considerably more protected from beta-lactams under wet-dry cycles with MSW phases, than they are under constantly wet conditions. This is due to the combined effect of several mechanisms, including tolerance triggered by inherent properties of MSW, i.e., high salt concentrations and slow cell growth, and the deactivation of antibiotics due to physicochemical properties of MSW. Remarkably, we also find evidence for a cross-protection effect, where addition of lethal doses of antibiotic before drying significantly increases cells’ survival under MSW. As wet-dry cycles with MSW and beta-lactams, as well as other antibiotics, are common in vast terrestrial microbial habitats, our findings are expected to have significant implications for how we understand antibiotic response, population dynamics, and interspecies interactions in these globally important microbial ecosystems.
TiPS: rapidly simulating trajectories and phylogenies from compartmental models
Authors: Danesh Gonché , Saulnier Emma , Gascuel Olivier , Choisy Marc , Alizon Samuel
Published: November 2020

We introduceTiPS, an R package to generate trajectories and phylogenetic trees associated with a compartmental model. Trajectories are simulated using Gillespie’s exact or approximate stochastic simulation algorithm, or a newly-proposed mixed version of the two. Phylogenetic trees are simulated from a trajectory under a backwards-in-time approach (i.e. coalescent).TiPSis based on theRcpppackage, allowing to combine the flexibility of R for model definition and the speed of C++ for simulations execution. The model is defined in R with a set of reactions, which allow capturing heterogeneity in life cycles or any sort of population structure.TiPSconverts the model into C++ code and compiles it into a simulator that is interfaced in Rviaa function. Furthermore, the package allows defining time periods in which the model’s parameters can take different values. This package is particularly well suited for population genetics and phylodynamics studies that need to generate a large number of phylogenies used for population dynamics studies. This package is available on the CRAN athttps://cran.r-project.org/package=TiPS.
Glymphatic function in the gyrencephalic brain
Authors: Bèchet Nicholas Burdon , Shanbhag Nagesh C. , Lundgaard Iben
Published: November 2020

Identification of the perivascular compartment as the point of exchange between cerebrospinal fluid (CSF) and interstitial fluid mediating solute clearance in the brain, named the glymphatic system, has emerged as an important clearance pathway for neurotoxic peptides such as amyloid-beta. However, the foundational science of the glymphatic system is based on rodent studies. Here we investigated whether the glymphatic system exists in a large mammal with a highly gyrified brain. CSF penetration into the brain via perivascular pathways, a hallmark of glymphatic function, was seen throughout the gyrencephalic cortex and subcortical structures, validating the conservation of the glymphatic system in a large mammal. Macroscopic CSF tracer distribution followed the sulci and fissures showing that the gyri enhance CSF dispersion. Three-dimensional renditions from light sheet microscopy showed that CSF influx through perivascular spaces was 4-fold more extensive in the pig brain than in mice. This demonstrates the existence of an advanced solute transport system in the gyrencephalic brain that could be utilised therapeutically for enhancing waste clearance.
Beneficent and maleficent effects of cations on bufadienolide binding to Na+,K+-ATPase
Authors: Ladefoged Lucy K. , Schiøtt Birgit , Fedosova Natalya U.
Published: November 2020

Kinetic properties and crystal structures of the Na+,K+-ATPase in complex with cardiotonic steroids (CTS) revealed significant differences between CTS subfamilies (Laursen et al., 2015): beneficial effects of K+on bufadienolide binding strongly contrasted with K+/cardenolide antagonism. To solve this riddle we applied docking and molecular dynamics simulations of the complexes involving Na+,K+-ATPase, bufadienolides (bufalin, cinobufagin), and ions (K+, Na+, Mg2+). The results revealed that bufadienolide binding is affected by i) electrostatic attraction of the lactone ring by a cation, and ii) the ability of a cation to stabilize and “shape” the site constituted by transmembrane helices of the α-subunit (αM1-6). The latter effect was due to varying coordination patterns involving amino acid residues from helix bundles αM1-4 and αM5-10. Substituents on the steroid core of a bufadienolide add to and modify the cation effects. The above rationale is fully consistent with the ion effects on the kinetics of Na+,K+-ATPase/bufadienolide interactions.
Effects of sea ice and wind speed on Phytoplankton spring bloom in Central and Southern Baltic Sea
Authors: Pärn Ove , Lessin Gennadi , Stips Adolf
Published: November 2020

In this study, the effects of sea ice and wind speed on the timing and composition of phytoplankton spring bloom in the central and southern Baltic Sea are investigated by a hydrodynamic–biogeochemical model and observational data. The modelling experiment compared the results of a reference run in the presence of sea ice with those of a run in the absence of sea ice, which confirmed that ecological conditions differed significantly for both the scenarios. It has been found that diatoms dominate the phytoplankton biomass in the absence of sea ice, whereas dinoflagellates dominate the biomass in the presence of thin sea ice. The study concludes that under moderate ice conditions (representing the last few decades), dinoflagellates dominate the spring bloom phytoplankton biomass in the Baltic Sea, whereas diatoms will be dominant in the future as a result of climate change i.e. in the absence of sea ice..
Soil microbial community responses to short-term nitrogen addition in China’s Horqin Sandy Land
Authors: Yayi Niu , Yulong Duan , Yuqiang Li , Xuyang Wang , Yun Chen , Lilong Wang
Published: November 2020

Anthropogenic nitrogen (N) addition has increased soil nutrient availability, thereby affecting ecosystem processes and functions in N-limited ecosystems. Long-term N addition decreases plant biodiversity, but the effects of short-term N addition on soil microbial community is poorly understood. The present study examined the impacts of short-term N addition (NH4NO3) on these factors in a sandy grassland and semi-fixed sandy land in the Horqin Sandy Land. We measured the responses of soil microbial biomass C and N; on soil β-1,4-glucosidase (BG) and β-1,4-N-acetylglucosaminidase (NAG) activity; and soil microflora characteristics to N additions gradient with 0 (control), 5 (N5), 10 (N10), and 15 (N15) g N m−2yr−1. The soil microbial biomass indices, NAG activity, and soil microflora characteristics did not differ significantly among the N levels, and there was no difference at the two sites. The competition for N between plants and soil microbes was not eliminated by short-term N addition due to the low soil nutrient and moisture contents, and the relationships among the original soil microbes did not change. However, N addition increased BG activity in the N5 and N10 additions in the sandy grassland, and in the N5, N10, and N15 additions in the semi-fixed sandy land. This may be due to increased accumulation and fixation of plant litter into soils in response to N addition, leading to increased microbial demand for a C source and increased soil BG activity. Future research should explore the relationships between soil microbial community and N addition at the two sites.
Target-Decoy MineR for determining the biological relevance of variables in noisy data sets
Authors: Ovando-Vázquez Cesaré , Cázarez-García Daniel , Winkler Robert
Published: November 2020

Machine learning algorithms excavate important variables from biologicalbig data. However, deciding on the biological relevance of identified variables is challenging. The addition of artificial noise, ‘decoy’ variables, to raw data, ‘target’ variables, enables calculating a false-positive rate (FPR) and a biological relevance probability (BRp) for each variable rank. These scores allow the setting of a cut-off for informative variables can be defined, depending on the required sensitivity/ specificity of a scientific question. We demonstrate the function of theTarget-Decoy MineR(TDM) with synthetic data and with experimental metabolomics results. The Target-Decoy MineR is suitable for different types of quantitative data in tabular format. An implementation of the algorithm in R is freely available fromhttps://bitbucket.org/cesaremov/targetdecoy_mining/.
Novel proteostasis reporter mouse reveals different effects of cytoplasmic and nuclear aggregates on protein quality control in neurons
Authors: Blumenstock Sonja , Schulz-Trieglaff Elena Katharina , Bolender Anna-Lena , Voelkl Kerstin , Lapios Paul , Lindner Jana , Hipp Mark S. , Hartl F. Ulrich , Klein Rüdiger , Dudanova Irina
Published: November 2020

The cellular protein quality control machinery is important for preventing protein misfolding and aggregation, and decline in protein homeostasis (proteostasis) is believed to play a crucial role in age-related neurodegenerative disorders. However, how proteostasis capacity of neurons changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice forin vivoanalysis of neuronal proteostasis. The mice express EGFP-fused firefly luciferase (Fluc), a conformationally unstable protein that requires chaperones for proper folding and sensitively reacts to proteotoxic stress by formation of intracellular Fluc-EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked impairment in proteostasis in tauopathy mice, but not in Huntington’s disease mice. Mechanistic investigations in primary neuronal cultures demonstrate that cytoplasmic, but not nuclear, aggregates cause defects of cellular protein quality control. Thus, the Fluc-EGFP reporter mice enable new insights into proteostasis alterations in different diseases.
Theta rhythmicity governs the timing of behavioral and hippocampal responses in humans specifically during memory-dependent tasks
Authors: Wal Marije ter , Domingo Juan Linde , Lifanov Julia , Roux Frederic , Kolibius Luca , Gollwitzer Stephanie , Lang Johannes , Hamer Hajo , Rollings David , Sawlani Vijay , Chelvarajah Ramesh , Staresina Bernhard , Hanslmayr Simon , Wimber Maria
Published: November 2020

1.

Memory formation and reinstatement are thought to lock to the hippocampal theta rhythm, predicting that encoding and retrieval processes appear rhythmic themselves. Here, we show that rhythmicity can be observed in behavioral responses from memory tasks, where participants indicate, using button presses, the timing of encoding or retrieval of cue-object associative memories. We found no evidence for rhythmicity in button presses for visual tasks using the same stimuli, or for questions about already retrieved objects. The oscillations for correctly remembered trials center in the slow theta frequency range (1-5 Hz), while responses from later forgotten trials do not lock to the behavioral oscillation. Using intracranial EEG recordings, we show that the memory task induces temporally extended phase consistency in hippocampal local field potentials at slow theta frequencies, but only for correctly remembered trials, providing a mechanistic underpinning for the theta oscillations found in behavioral responses.
Oligodendrocyte secreted factors shape hippocampal GABAergic neuron transcriptome and physiology
Authors: Mazuir Elisa , Richevaux Louis , Nassar Merie , Robil Noémie , de la Grange Pierre , Lubetzki Catherine , Fricker Desdemona , Sol-Foulon Nathalie
Published: November 2020

Oligodendrocytes form myelin for central nervous system axons and release factors which signal to neurons during myelination. Here, we ask how oligodendroglial factors influence hippocampal GABAergic neuron physiology. In mixed hippocampal cultures GABAergic neurons fired action potentials of short duration and received high frequencies of excitatory synaptic events. In purified neuronal cultures without glial cells, GABAergic neuron excitability increased and the frequency of synaptic events decreased. These effects were largely reversed by adding oligodendrocyte conditioned medium. We compared the transcriptomic signature with the electrophysiological phenotype of single neurons in these three culture conditions. Genes expressed by single pyramidal or GABAergic neurons largely conformed to expected cell-type specific patterns. Multiple genes of GABAergic neurons were significantly downregulated by the transition from mixed cultures containing glial cells to purified neuronal cultures. Levels of these genes were restored by the addition of oligodendrocyte conditioned medium to purified cultures. Clustering genes with similar changes in expression between different culture conditions revealed processes affected by oligodendroglial factors. Enriched genes are linked to roles in synapse assembly, action potential generation and transmembrane ion transport, including of zinc. These results provide new insight into the molecular targets by which oligodendrocytes influence neuron excitability and synaptic function.
Human antigen-specific memory natural killer cell responses develop against HIV-1 and influenza virus and are dependent on MHC-E restriction
Authors: Jost Stephanie , Lucar Olivier , Yoder Taylor , Kroll Kyle , Sugawara Sho , Smith Scott , Jones Rhianna , Tweet George , Werner Alexandra , Tomezsko Phillip J. , Dugan Haley L. , Ghofrani Joshua , Altfeld Marcus , Grundhoff Adam , Muller-Trutwin Michaela , Goepfert Paul , Reeves R. Keith
Published: November 2020

For over a decade, multiple studies have disputed the notion of natural killer (NK) cells as purely innate lymphocytes by demonstrating that they are capable of putative antigen-specific immunological memory against multiple infectious agents including two critical global health priorities – HIV and influenza. However, the mechanisms underlying antigen specificity remain unknown. Herein, we demonstrate that antigen-specific human NK cell memory develops upon exposure to both HIV and influenza, unified by a conserved and epitope-specific targetable mechanism firmly dependent on the activating CD94/NKG2C receptor and its ligand HLA-E, and confirm these findings by three rigorous and novel assays. We validated the permanent acquisition of antigen-specificity by individual memory NK cells by single-cell cloning. We identified biomarkers of antigen-specific NK cell memory through RNA-Seq transcriptomic fingerprints and complex immunophenotyping by 30-parameter flow cytometry showing elevated expression of KLRG1, α4β7 and NKG2C. Finally, we show individual HLA-E-restricted peptides that may constitute the dominant response in HIV-1- and influenza-infected persons in vivo. Our findings clarify the mechanisms behind formation of antigen-specific memory NK cells, and suggest they could be targeted for future vaccines, cure strategies, or other therapeutic interventions.
Evolution of the genetic architecture of local adaptations under genetic rescue is determined by mutational load and polygenicity
Authors: Zhang Yulin , Stern Aaron J. , Nielsen Rasmus
Published: November 2020

Inbred populations often suffer from heightened mutational load and decreased fitness due to lower efficiency of purifying selection at small effective population size. Genetic rescue (GR) is a tool that is studied and deployed with the aim of increasing fitness of such inbred populations. The success of GR is known to depend on certain factors that may vary between different populations, such as their demographic history and distribution of dominance effects of mutations. While we understand the effects of these factors on the evolution of overall ancestry in the inbred population after GR, it is less clear what the effect is on local adaptations and their genetic architecture. To this end, we conduct a population genetic simulation study evaluating the effect of several different factors on the efficacy of GR including trait complexity (Mendelian vs. polygenic), dominance effects, and demographic history. We find that the effect on local adaptations depends highly on the mutational load at the time of GR, which is shaped dynamically by interactions between demographic history and dominance effects of deleterious variation. While local adaptations are generally restored post-GR in the long run, in the short term they are often compromised in the process of purging deleterious variation. We also show that while local adaptations are almost always fully restored, the degree to which ancestral genetic variation comprising the trait is replaced by donor variation can vary drastically, and is especially high for complex traits. Our results provide considerations for practical GR and its effects on trait evolution.
Evolution of Natural Lifespan Variation and Molecular Strategies of Extended Lifespan
Authors: Kaya Alaattin , Phua Cheryl Zi Jin , Lee Mitchell , Wang Lu , Tyshkovskiy Alexander , Ma Siming , Barre Benjamin , Liu Weiqiang , Harrison Benjamin R. , Zhao Xiaqing , Zhou Xuming , Wasko Brian M. , Bammler Theo K. , Promislow Daniel E. , Kaeberlein Matt , Gladyshev Vadim N.
Published: November 2020

To understand the genetic basis and selective forces acting on longevity, it is useful to examine lifespan variation among closely related species, or ecologically diverse isolates of the same species, within a controlled environment. In particular, this approach may lead to understanding mechanisms underlying natural variation in lifespan. Here, we analyzed 76 ecologically diverse wild yeast isolates and discovered a wide diversity of replicative lifespan. Phylogenetic analyses pointed to genes and environmental factors that strongly interact to modulate the observed aging patterns. We then identified genetic networks causally associated with natural variation in replicative lifespan across wild yeast isolates, as well as genes, metabolites and pathways, many of which have never been associated with yeast lifespan in laboratory settings. In addition, a combined analysis of lifespan-associated metabolic and transcriptomic changes revealed unique adaptations to interconnected amino acid biosynthesis, glutamate metabolism and mitochondrial function in long-lived strains. Overall, our multi-omic and lifespan analyses across diverse isolates of the same species shows how gene-environment interactions shape cellular processes involved in phenotypic variation such as lifespan.
What Markov state models can and cannot do: Correlation versus path-based observables in protein folding models
Authors: Suárez Ernesto , Wiewiora Rafal P. , Wehmeyer Chris , Noé Frank , Chodera John D. , Zuckerman Daniel M.
Published: November 2020

Markov state models (MSMs) have been widely applied to study the kinetics and pathways of protein conformational dynamics based on statistical analysis of molecular dynamics (MD) simulations. These MSMs coarse-grain both configuration space and time in ways that limit what kinds of observables they can reproduce with high fidelity over different spatial and temporal resolutions. Despite their popularity, there is still limited understanding of which biophysical observables can be computed from these MSMs in a robust and unbiased manner, and which suffer from the space-time coarse-graining intrinsic in the MSM model. Most theoretical arguments and practical validity tests for MSMs rely on long-time equilibrium kinetics, such as the slowest relaxation timescales and experimentally observable time-correlation functions. Here, we perform an extensive assessment of the ability of well-validated protein folding MSMs to accuractely reproduce path-based observable such as mean first-passage times (MFPTs) and transition path mechanisms compared to a direct trajectory analysis. We also assess a recently proposed class of history-augmented MSMs (haMSMs) that exploit additional information not accounted for in standard MSMs. We conclude with some practical guidance on the use of MSMs to study various problems in conformational dynamics of biomolecules. In brief, MSMs can accurately reproduce correlation functions slower than the lag time, but path-based observables can only be reliably reproduced if the lifetimes of states exceed the lag time, which is a much stricter requirement. Even in the presence of short-lived states, we find that haMSMs reproduce path-based observables more reliably.
Bulk flow of cerebrospinal fluid observed in periarterial spaces is not an artifact of injection
Authors: Raghunandan Aditya , Ladrón-de-Guevara Antonio , Tithof Jeffrey , Mestre Humberto , Nedergaard Maiken , Thomas John H. , Kelley Douglas H.
Published: November 2020

Cerebrospinal fluid (CSF) flowing through periarterial spaces is integral to the brain’s mechanism for clearing metabolic waste products. Experiments that track tracer particles injected into the cisterna magna of mouse brains have shown evidence of pulsatile CSF flow in pial periarterial spaces, with a bulk flow in the same direction as blood flow. However, the driving mechanism remains elusive. Several studies have suggested that the bulk flow might be an artifact, driven by the injection itself. Here, we address this hypothesis with newin vivoexperiments where tracer particles are injected into the cisterna magna using a dual-syringe system, with simultaneous injection and withdrawal of equal amounts of fluid. This method produces no net increase in CSF volume and no significant increase in intracranial pressure. Yet, particle-tracking reveals flows in the pial periarterial spaces that are completely consistent with the flows observed in earlier experiments with single-syringe injection.
The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways
Authors: Laporte Manon , Stevaert Annelies , Raeymaekers Valerie , Van Berwaer Ria , Martens Katleen , Pöhlmann Stefan , Naesens Lieve
Published: November 2020

The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV-1 and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. First, the SARS-CoV-2 spike (SARS-2-S) reached higher levels in pseudoparticles when produced at 33°C instead of 37°C. Even stronger preference for the upper airway temperature of 33°C was evident for the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV-1 and MERS-CoV favored 37°C, in accordance with their preference for the lower airways. Next, SARS-2-S proved efficiently activated by TMPRSS13, besides the previously identified host cell protease TMPRSS2, which may broaden the cell tropism of SARS-CoV-2. TMPRSS13 was found to be an effective spike activator for the virulent coronaviruses but not the common cold HCoV-229E virus. Activation by these proteases requires pre-cleavage of the SARS-2-S S1/S2 cleavage loop, and both its furin motif and extended loop length proved critical to achieve virus entry into airway epithelial cells. Finally, we show that the D614G mutation in SARS-2-S increases S protein stability and expression at 37°C, and promotes virus entry via cathepsin B/L activation. These spike properties might promote virus spread, potentially explaining why the G614 variant is currently predominating worldwide. Collectively, our findings indicate how the coronavirus spike protein is fine-tuned towards the temperature and protease conditions of the airways, to enhance virus transmission and pathology.

SIGNIFICANCE STATEMENT

The rapid spread of SARS-CoV-2, the cause of COVID-19, is related to abundant replication in the upper airways, which is not observed for other highly pathogenic human coronaviruses. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. Coronavirus spikes exhibit distinct temperature preference to precisely match the upper (~33°C) or lower (37°C) airways. We identified airway proteases that activate the spike for virus entry into cells, including one protease that may mediate coronavirus virulence. Also, a link was seen between spike stability and entry via endosomal proteases. This mechanism of spike fine-tuning could explain why the SARS-CoV-2 spike-D614G mutant is more transmissible and therefore globally predominant.
Subsidy Accessibility Drives Asymmetric Food Web Responses
Authors: Gutgesell Marie , McMeans Bailey C. , Guzzo Matthew M. , deGroot Valesca , Fisk Aaron T. , Johnson Timothy B. , McCann Kevin S.
Published: November 2020

Global change is fundamentally altering flows of natural and anthropogenic subsidies across space and time. After a pointed call for research on subsidies in the 1990s, an industry of empirical work has documented the ubiquitous role subsidies play in ecosystem structure, stability and function. Here, we argue that physical constraints (e.g., water temperature) and species traits can govern a species’ accessibility to resource subsidies, and that these physical constraints have been largely overlooked in the subsidy literature. We examined the input of a high quality, point-source anthropogenic subsidy into a recipient freshwater lake food web (i.e., released net-pen aquaculture feed in Parry Sound, Lake Huron), to demonstrate the importance of subsidy accessibility in governing recipient whole food web responses. By using a combined bio-tracer approach, we detect a gradient in accessibility of the anthropogenic subsidy within the surrounding food web driven by the thermal tolerances of three constituent species. This thermally-driven accessibility gradient drives asymmetrical changes in food web structure, effectively rewiring the recipient lake food web and altering patterns in secondary production with yet unknown stability consequences. Since aquaculture is predicted to increase significantly in coming decades to support growing human populations, and global change is altering temperature regimes, then this form of food web alteration may be expected to occur frequently. We argue that subsidy accessibility is a key characteristic of recipient food web interactions that must be considered when trying to understand the impacts of subsidies on ecosystem stability and function under continued global change.
Essential role of non-vesicular lipid transport in microtubule-controlled cell polarisation
Authors: Hersberger-Trost M. , Dreher D. , Huisman S.M. , Kijowski A.R. , Gemünden M. , Klemm R.W. , Brunner D.
Published: November 2020

Cell polarisation is a fundamental biological process. Fission yeast is a key model system to study the molecular basis of microtubule-controlled cell polarisation. In this process, cells define prospective growth sites by generating distinct plasma membrane domains enriched inde novosynthesised sterols. Microtubules restrict the number and location of these domains by depositing factors at the cell poles. The mechanisms underlying such sterol-rich membrane domain formation and polarisation are largely unknown. We found that the oxysterol-binding proteins kes1p, osh2p and kes3p define three independent sterol delivery pathways to the plasma membrane. These mediate different phases of cell polarisation in a phosphoinositide-dependent fashion and differ in their requirement for vesicular trafficking steps. The redundant, kes1p- and osh2p-dependent pathways are vital and prime cell polarisation by mediating the formation of randomly distributed sterol-rich plasma membrane domains. Subsequent microtubule-controlled polarisation of these domains preferentially employs kes1p that directly delivers sterols to the plasma membrane independent of cdc42p. In cells lacking kes1p, polarisation becomes cdc42p-dependent, utilising mainly the kes3p-dependent pathway. Our study uncovers an essential biological function for non-vesicular lipid transport and establishes a molecular basis for different sterol-delivery pathways acting in cdc42p-independent and cdc42p-dependent cell polarisation.
Neutrophil extracellular traps induce the epithelial-mesenchymal transition: implications in post-COVID-19 fibrosis
Authors: Pandolfi Laura , Bozzini Sara , Frangipane Vanessa , Percivalle Elena , De Luigi Ada , Violatto Martina Bruna , Lopez Gianluca , Gabanti Elisa , Carsana Luca , D’Amato Maura , Morosini Monica , De Amici Mara , Nebuloni Manuela , Fossali Tommaso , Colombo Riccardo , Saracino Laura , Codullo Veronica , Gnecchi Massimiliano , Bigini Paolo , Baldanti Fausto , Lilleri Daniele , Meloni Federica
Published: November 2020

The release of neutrophil extracellular traps (NETs), a process termed NETosis, avoids pathogen spread but may cause tissue injury. NETs have been found in severe COVID-19 patients, but their role in disease development is still unknown. The aim of this study is to assess the capacity of NETs to drive epithelial-mesenchymal transition (EMT) of lung epithelial cells and to analyze the involvement of NETs in COVID-19.

Neutrophils activated with PMA (PMA-Neu), a stimulus known to induce NETs formation, induce both EMT and cell death in the lung epithelial cell line, A549. Notably, NETs isolated from PMA-Neu induce EMT without cell damage. Bronchoalveolar lavage fluid of severe COVID-19 patients showed high concentration of NETs. Thus, we tested in anin vitroalveolar model the hypothesis that virus-induced NET may drive EMT. Co-culturing A549 at air-liquid interface with alveolar macrophages, neutrophils and SARS-CoV2, we demonstrated a significant induction of the EMT in A549 together with high concentration of NETs, IL8 and IL1β, best-known inducers of NETosis. Lung tissues of COVID-19 deceased patients showed that epithelial cells are characterized by increased mesenchymal markers. These results show for the first time that NETosis plays a major role in triggering lung fibrosis in COVID-19 patients.
StableDNMT3LOverexpression in SH-SY5Y Neurons Recreates a Facet of the Genome-Wide Down Syndrome DNA Methylation Signature
Authors: Laufer Benjamin I. , Gomez J. Antonio , Jianu Julia M. , LaSalle Janine M.
Published: November 2020

Down syndrome (DS) is characterized by a genome-wide profile of differential DNA methylation that is skewed towards hypermethylation in most tissues, including brain. The molecular mechanisms involve the overexpression of genes related to DNA methylation on chromosome 21. Here, we stably overexpressed the chromosome 21 gene DNA methyltransferase 3L (DNMT3L) in the human SH-SY5Y neuroblastoma cell line and assayed DNA methylation at over 26 million CpGs by whole genome bisulfite sequencing at three different developmental phases (undifferentiated, differentiating, and differentiated).DNMT3Loverexpression resulted in global CpG and CpG island hypermethylation as well as thousands of differentially methylated regions (DMRs). TheDNMT3LDMRs were skewed towards hypermethylation and mapped to genes involved in neurodevelopment, cellular signaling, and gene regulation. Merging the DMRs into a consensus profile where the cell lines clustered by genotype and then phase demonstrated that different regions of common genes are affected. The hypermethylated DMRs from all pairwise comparisons were enriched for regions of bivalent chromatin marked by H3K4me3 as well as differentially methylated CpGs from previous DS studies of diverse tissues. In contrast, the hypomethylated DMRs from all pairwise comparisons displayed a tissue-specific profile enriched for regions of heterochromatin marked by H3K9me3 during embryonic development. Taken together, we propose a mechanism whereby regions of bivalent chromatin that lose H3K4me3 during development are targeted by excess DNMT3L and become hypermethylated, while excess DNMT3L also evicts DNMT3A from heterochromatin, resulting in hypomethylation. Overall, these findings demonstrate thatDNMT3Loverexpression during neurodevelopment recreates a facet of the DS DNA methylation signature.
Potentiation of Long-Acting β2-Agonist and Glucocorticoid Responses in Human Airway Epithelial Cells by Modulation of Intracellular cAMP
Authors: Kim Yechan , Hou Vincent , Huff Ryan D. , Aguiar Jennifer A. , Revill Spencer , Tiessen Nicholas , Cao Quynh , Miller Matthew S. , Inman Mark D. , Ask Kjetil , Doxey Andrew C. , Hirota Jeremy A.
Published: November 2020

Introduction

Over 300 million people in the world live with asthma, resulting in 500,000 annual global deaths with future increases expected. It is estimated that around 50-80% of asthma exacerbations are due to viral infections. Currently, a combination of long-acting beta agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma, however it is still sub-optimal in 35-50% of moderate-severe asthmatics resulting in persistent lung inflammation, impairment of lung function, and risk of mortality. Mechanistically, LABA/GCS combination therapy results in synergistic efficacy mediated by intracellular cyclic adenosine monophosphate (cAMP).

Hypothesis

Increasing intracellular cAMP during LABA/GCS combination therapy via inhibiting phosphodiesterase 4 (PDE4) and/or blocking export of cAMP by ATP Binding Cassette Transporter C4 (ABCC4), will potentiate anti-inflammatory responses of mainstay LABA/GCS therapy.

Methods

Expression and localization experiments were performed usingin situhybridization and immunohistochemistry in human lung tissue from healthy subjects, while confirmatory transcript and protein expression analyses were performed in primary human airway epithelial cells and cell lines. Intervention experiments were performed on the human airway epithelial cell line, HBEC-6KT, by pre-treatment with combinations of LABA/GCS with PDE4 and/or ABCC4 inhibitors followed by Poly I:C or imiquimod challenge as a model for viral stimuli. Cytokine readouts for IL-6, IL-8, CXCL10/IP-10, and CCL5/RANTES were quantified by ELISA.

Results

Using archived human lung and human airway epithelial cells, ABCC4 gene and protein expression were confirmedin vitroandin situ. LABA/GCS attenuation of Poly I:C or imiquimod-induced IL-6 and IL-8 was potentiated with ABCC4 and PDE4 inhibition, which was greater when ABCC4 and PDE4 inhibition was combined. Modulation of cAMP levels had no impact on LABA/GCS modulation of Poly I:C-induced CXCL10/IP-10 or CCL5/RANTES.

Conclusion

Modulation of intracellular cAMP levels by PDE4 or ABCC4 inhibition is able to potentiate LABA/GCS efficacy in human airway epithelial cells challenged with viral stimuli. The data suggests further exploration of the value of adding cAMP modulators to mainstay LABA/GCS therapy in asthma for potentiated anti-inflammatory efficacy.