bluuebunny/crossref_metadata_2025 huggingface dataset:
Bayesian prediction of multivariate ecology from phenotypic data yields new insights into the diets of extant and extinct taxa
Authors: Wisniewski Anna L. , Nations Jonathan A. , Slater Graham J.
Published: May 2022

Morphology often reflects ecology, enabling the prediction of ecological roles for taxa that lack direct observations such as fossils. In comparative analyses, ecological traits, like diet, are often treated as categorical, which may aid prediction and simplify analyses but ignores the multivariate nature of ecological niches. Futhermore, methods for quantifying and predicting multivariate ecology remain rare. Here, we ranked the relative importance of 13 food items for a sample of 88 extant carnivoran mammals, and then used Bayesian multilevel modeling to assess whether those rankings could be predicted from dental morphology and body size. Traditional diet categories fail to capture the true multivariate nature of carnivoran diets, but Bayesian regression models derived from living taxa have good predictive accuracy for importance ranks. Using our models to predict the importance of individual food items, the multivariate dietary niche, and the nearest extant analogs for a set of data-deficient extant and extinct carnivoran species confirms long-standing ideas for some taxa, but yields new insights about the fundamental dietary niches of others. Our approach provides a promising alternative to traditional dietary classifications. Importantly, this approach need not be limited to diet, but serves as a general framework for predicting multivariate ecology from phenotypic traits.
Condensation of the fusion focus by the intrinsically disordered region of the formin Fus1 is essential for cell-cell fusion
Authors: Billault-Chaumartin Ingrid , Muriel Olivia , Michon Laetitia , Martin Sophie G
Published: May 2022

Spatial accumulation of secretory vesicles underlies various cellular processes, such as neurotransmitter release at neuronal synapses [1], hyphal steering in filamentous fungi [2, 3], and local cell wall digestion preceding the fusion of yeast gametes [4]. Secretory vesicles transported on actin filaments by myosin V motors form clusters that serve as pool for local content release. During fission yeast Schizosaccharomyces pombe gamete fusion, the actin fusion focus assembled by the formin Fus1 concentrates secretory vesicles carrying cell wall digestive enzymes [5-7]. Focus position and coalescence are controlled by local signalling and actin-binding proteins to prevent inappropriate cell wall digestion that would cause lysis [6, 8-10], but the mechanisms of focusing have been elusive. Here, we show that the regulatory N-terminus of Fus1 contains an intrinsically disordered region (IDR) that mediates Fus1 condensation in vivo and forms dense assemblies that exclude other macromolecules. Fus1 lacking its IDR fails to condense in a tight focus and causes cell lysis during attempted cell fusion. Remarkably, replacement of Fus1 IDR with a heterologous low-complexity region that forms liquid condensates fully restores Fus1 condensation and function. By contrast, replacement of Fus1 IDR with a domain that forms more stable oligomers restores condensation but poorly supports cell fusion, suggesting that condensation is tuned to yield a structure selectively permeable for secretory vesicles. We propose that condensation of actin structures by an intrinsically disordered region may be a general mechanism for actin network organisation and the selective local concentration of secretory vesicles.
Insights into Long-term Acclimation Strategies of Grapevines in Response to Multi-decadal Cyclical Drought
Authors: Nagahatenna Dilrukshi S. K. , Furlan Tarita S. , Edwards Everard J. , Ramesh Sunita A. , Pagay Vinay
Published: May 2022

The Australian wine industry is currently under pressure to sustain its profitability due to climate change. Therefore, there is a pressing need to explore grapevine genetic diversity and identify superior clones with improved drought resistance. We previously characterised more than 15,000 dry-farmed (for over 65 years) Cabernet Sauvignon clones in a vineyard and identified three drought-tolerant (DT) clones, which can maintain significantly higher intrinsic water use efficiency (WUEi) under limited soil moisture than drought-sensitive (DS) clones. To understand whether DT clones grown under multi-decadal cyclical drought can prime their vegetatively-propagated clonal progenies for future drought events, in this study, all DT and DS vegetative progenies were propagated with commercial clones in the glasshouse. Their physiological and molecular responses were investigated under well-watered and two recurrent drought (D1 and D2) conditions. We observed that concentration of a natural priming agent, γ-amino butyric acid (GABA), were significantly higher in all DT progenies relative to other progenies under drought. Both commercial and DT progenies exhibited improved gas exchange, photosynthetic performance and WUEi under recurrent drought events relative to DS progenies. Our results suggest that DT progenies have adapted to be in a “primed state” to withstand future drought events.
Autonomous untethered microinjectors for gastrointestinal delivery of insulin
Authors: Ghosh Arijit , Liu Wangqu , Li Ling , Pahapale Gayatri , Choi Si Young , Xu Liyi , Huang Qi , Selaru Florin M. , Gracias David H.
Published: May 2022

The delivery of macromolecular drugs via the gastrointestinal (GI) tract is challenging. Macromolecular drugs display low stability and poor absorption across the intestinal epithelium. While permeation-enhancing drug delivery methods can increase the bioavailability of low molecular weight drugs, the effective delivery of high molecular weight drugs across the tight epithelial cell junctions remains a formidable challenge. Here, we describe autonomous microinjectors that can efficiently penetrate the GI mucosa and deliver insulin systemically. In addition, we performed in vitro studies to characterize insulin release and the penetration capacity of microinjectors and measure in vivo release of insulin in live rats. We found that the microinjectors administered within the luminal GI tract could deliver insulin trans-mucosally to the systemic circulation at similar levels to intravenously administered insulin. Due to their small size, tunability in sizing and dosing, wafer-scale fabrication, and parallel, autonomous operation, we anticipate that these novel microinjectors could significantly advance drug delivery across the GI tract mucosa to the systemic circulation.
Physical limits to acceleration of chemical reactions inside phase-separated compartments
Authors: Schmit Jeremy D. , Michaels Thomas C. T.
Published: May 2022

We present a theoretical analysis of phase separated compartments as a means to facilitate chemical reactions. We find that the attractive interactions that concentrate reactants within the dense phase inhibit reactions by lowering the chemical potential and mobility of the reactants. Therefore, condensed phases are only beneficial if mobility in the condensed phase can be maintained. This can be achieved in multi-step reactions, where the proximity between enzymatic steps results in higher efficiency with less unreacted substrate, but does not increase the reaction rate. Alternatively, mobility can be maintained if recruitment to the condensed phase is driven by multiple attractive moieties that can bind and release independently. However, the spacers necessary to ensure independence between stickers are prone to entangle with the dense phase scaffold. We find an optimal sticker affinity that balances the need for rapid binding/unbinding kinetics and minimal entanglement. Reaction rates can be accelerated by shrinking the size of the dense phase with a corresponding increase in the number of stickers to enhance recruitment.
The iron-sulfur cluster is critical for DNA binding by human DNA polymerase ε
Authors: Lisova Alisa E. , Baranovskiy Andrey G. , Morstadt Lucia M. , Babayeva Nigar D. , Stepchenkova Elena I. , Tahirov Tahir H.
Published: May 2022

DNA polymerase ε (Polε) is a key enzyme for DNA replication in eukaryotes. It is attached to a helicase and performs DNA synthesis on the leading strand. Recently it was shown that the catalytic domain of yeast Polε (PolεCD) contains a [4Fe-4S] cluster located at the base of the processivity domain (P-domain) and coordinated by four conserved cysteines. In this work, we have shown that human PolεCD (hPolεCD) expressed in bacterial cells also contains an iron-sulfur cluster. In comparison, recombinant hPolεCD produced in insect cells contains an eight-fold-lower level of iron. Interestingly, the iron content correlates with the level of DNA-binding molecules, which suggests an important role of the iron-sulfur cluster in hPolε interaction with DNA. Indeed, mutation of two conserved cysteines that coordinate the cluster abolished template:primer binding and, therefore, DNA polymerase and proofreading exonuclease activities. We propose that the cluster regulates the conformation of the P-domain, which, like a gatekeeper, controls access to a DNA-binding cleft for a template:primer. In addition, we performed kinetic and binding studies of hPolεCD. The binding studies demonstrated low affinity of hPolεCD to DNA and a strong effect of salt concentration on stability of the hPolεCD/DNA complex. Pre-steady-state kinetic studies have shown a maximal polymerization rate constant of 51.5 s-1 and a relatively low affinity to incoming dNTP with an apparent KD of 105 μM. This work provides notable insight into the role of a [4Fe-4S] cluster in Polε function.
Temporal analysis suggests a reciprocal relationship between 3D chromatin structure and transcription
Authors: Reed Kathleen S. M. , Davis Eric S. , Bond Marielle L. , Cabrera Alan , Thulson Eliza , Quiroga I. Yoseli , Cassel Shannon , Woolery Kamisha T. , Hilton Isaac , Won Hyejung , Love Michael I. , Phanstiel Douglas H.
Published: May 2022

To infer potential causal relationships between 3D chromatin structure, enhancers, and gene transcription, we mapped each feature in a genome-wide fashion across eight narrowly-spaced timepoints of macrophage activation. Enhancers and genes connected by loops exhibited stronger correlations between histone H3K27 acetylation and expression than can be explained by genomic distance or physical proximity alone. Changes in acetylation at looped distal enhancers preceded changes in gene expression. Changes in gene expression exhibit a directional bias at differential loop anchors; gained loops are associated with increased expression of genes oriented away from the center of the loop, while lost loops were often accompanied by high levels of transcription with the loop boundaries themselves. Taken together, these results are consistent with a reciprocal relationship in which loops can facilitate increased transcription by connecting promoters to distal enhancers while high levels of transcription can impede loop formation.

HIGHLIGHTS

LPS + IFNγ triggers genome-wide changes in chromatin looping, enhancer acetylation, and gene expression

Looped enhancer-promoter pairs exhibit ordered and correlated changes in acetylation and expression

Changes in gene expression exhibit a directional bias at differential loop anchors

Lost loops are associated with high levels of transcription within loop boundaries
Truncated radial glia as a common precursor in the late corticogenesis of gyrencephalic mammals
Authors: Bilgic Merve , Wu Quan , Suetsugu Taeko , Shitamukai Atsunori , Tsunekawa Yuji , Shimogori Tomomi , Kadota Mitsutaka , Nishimura Osamu , Kuraku Shigehiro , Kiyonari Hiroshi , Matsuzaki Fumio
Published: May 2022

The diversity of neural stem cells is a hallmark of the cerebral cortex development in gyrencephalic mammals, such as Primates and Carnivora. Among them, ferrets are a good model for mechanistic studies. However, information on their neural progenitor cells (NPC), termed radial glia (RG), is limited. Here, we surveyed the temporal series of single-cell transcriptomes of progenitors regarding ferret corticogenesis and, found a conserved diversity and temporal trajectory between human and ferret NPC, despite the large timescale difference. We found truncated RG (tRG) in ferret cortical development, a progenitor subtype previously described in humans. The combination ofin silicoandin vivoanalyses identified that tRG differentiate into both ependymal and astrogenic cells. Via transcriptomic comparison, we predict that this is also the case in humans. Our findings suggest that tRG plays a role in the formation of adult ventricles, thereby providing the architectural bases for brain expansion.
Development of recombinant monoclonal antibodies targeting conserved VlsE epitopes in Lyme disease pathogens
Authors: Li Li , Di Lia , Akther Saymon , Zeglis Brian M. , Qiu Weigang
Published: May 2022

VlsE (variable major protein-likesequence,expressed) is an outer surface protein of the Lyme disease pathogen (Borreliellaspecies) and a key diagnostic biomarker of Lyme disease. However, the high sequence variability of VlsE poses a challenge to the development of consistent VlsE-based diagnostics and therapeutics. In addition, the standard diagnostic protocols detect immunoglobins elicited by the Lyme pathogen, not the presence of pathogen or its derived antigens. Here we describe the development of recombinant monoclonal antibodies (rMAbs) that bind specifically to conserved epitopes on VlsE. We first quantified amino-acid sequence variability encoded by thevlsgenes from thirteenB. burgdorferigenomes by evolutionary analyses. We showed broad inconsistencies of the sequence phylogeny with the genome phylogeny, indicating rapid gene duplications, losses, and recombination at thevlslocus. To identify conserved epitopes, we synthesized peptides representing five long conserved invariant regions (IRs) on VlsE. We tested the antigenicity of these five IR peptides using sera from three mammalian host species including human patients, the natural reservoir white-footed mouse (Peromyscus leucopus), and VlsE-immunized New Zealand rabbits (Oryctolagus cuniculus). The IR4 and IR6 peptides emerged as the most antigenic and reacted strongly with both the human and rabbit sera, while all IR peptides reacted poorly with sera from natural hosts. Four rMAbs binding specifically to the IR4 and IR6 peptides were identified, cloned, and purified. Given their specific recognition of the conserved epitopes on VlsE, these IR-specific rMAbs are promising diagnostic and theragnostic agents for direct detection of Lyme disease pathogens regardless of strain heterogeneity.
Polyimidazolium protects against an invasive clinical isolate of Salmonella Typhimurium
Authors: Mon Khin K. Z. , Si Zhangyong , Chan-Park Mary B. , Kenney Linda J.
Published: May 2022

Frequent outbreaks of Salmonella Typhimurium infection in both the animal and human population with potential for zoonotic transmission pose a significant threat to the public health sector. The rapid emergence and spread of more invasive multidrug-resistant clinical isolates of Salmonella further highlight the need for the development of new drugs with effective broad-spectrum bactericidal activities. Synthesis and evaluation of main-chain cationic polyimidazolium 1 (PIM1) against several gram-positive and gram-negative bacteria have previously demonstrated the efficacy profile of PIM1. The present study focuses on antibacterial and anti-biofilm activities of PIM1 against Salmonella both in vitro and in ovo setting. In vitro, PIM1 exhibited bactericidal activity against all tested three strains of Salmonella at a low dosage of 8 μg/ml. Anti-biofilm activity of PIM1 was evident with complete inhibition for the initial attachment of biofilms at 16 μg/ml and degradation of pre-formed biofilms in a dose-dependent manner. During the host cell infection process, PIM1 reduces extracellular bacterial adhesion and invasion rates to limit the establishment of infection. Once intracellular, the drug-resistant strain was tolerant and protected from PIM1 treatment. In a chicken egg infection model, PIM1 exhibited therapeutic activity for both Salmonella strains with stationary-phase and exponential-phase inocula. Moreover, PIM1 showed a remarkable efficacy against the stationary phase inocula of drug-resistant Salmonella by eliminating the bacteria burden in >50% of infected chicken egg embryos. Collectively, PIM1 has demonstrated its potential as a drug candidate for treatment of Salmonella infections, as well as a solution to tackle egg contamination issues on poultry farms.
Acinetobacter baumannii and cefiderocol between cidality and adaptability
Authors: Stracquadanio Stefano , Bonomo Carmelo , Marino Andrea , Bongiorno Dafne , Privitera Grete Francesca , Bivona Dalida Angela , Mirabile Alessia , Bonacci Paolo Giuseppe , Stefani Stefania
Published: May 2022

Among the bacterial species included in the ESKAPE group, Acinetobacter baumannii is of great interest due to its intrinsic and acquired resistance to many antibiotic classes and its ability to infect different body districts. Cefiderocol is a novel cephalosporin active against Gram-negative bacteria with promising efficacy on A. baumannii infections, but some studies have reported therapeutic failures even in the presence of susceptible A. baumannii strains.

This study aims to investigate the interactions between cefiderocol and ten A. baumannii strains with different susceptibility profiles to this drug. We confirmed diverse susceptibility profiles in the strains, with resistance values close to the EUCAST-proposed breakpoints. MBC/MIC (minimal bactericidal concentration/minimal inhibitory concentration) ratios, demonstrated bactericidal activity of the drug; on the other hand, bacterial regrowth was evident after exposition to cefiderocol, as were changes in the shape of colonies and bacterial cells. A switch to a non-susceptible phenotype in the presence of high cefiderocol concentrations was found as adaptation mechanisms implemented by these A. baumannii strains to overcome the cidal activity of this antibiotic, also confirmed by the presence of heteroresistant, unstable subpopulations. As our isolates harbored numerous β-lactamase genes, β-lactamase inhibitors showed the ability to restore the antimicrobial activity of cefiderocol regardless of the different non-susceptibility levels of the tested strains.

These in vitro results, can sustain the concept of using combination therapy to eliminate drug-adapted subpopulations and regain full cefiderocol activity in this difficult-to-treat species.
Bacterial association with metals enables in vivo tracking of microbiota using magnetic resonance imaging
Authors: Donnelly Sarah C. , Gelman Neil , Thompson R. Terry , Prato Frank S. , Burton Jeremy P. , Goldhawk Donna E.
Published: May 2022

Bacteria constitute a significant part of the biomass of the human microbiota, but their interactions are complex and difficult to replicate outside the host. Exploiting the superior resolution of magnetic resonance imaging (MRI) to examine signal parameters of selected human isolates may allow tracking of their dispersion throughout the body. We investigated longitudinal and transverse MRI relaxation rates and found significant differences between several bacterial strains. Common commensal strains of lactobacilli display notably high MRI relaxation rates, partially explained by outstanding cellular manganese content, while other species contain more iron than manganese. Lactobacillus crispatus show particularly high values, 4-fold greater than any other species; over 10-fold greater signal than relevant tissue background; and a linear relationship between relaxation rate and fraction of live cells. Different bacterial strains have detectable, repeatable MRI relaxation rates that in future may enable tracking of their persistence in the human body for enhanced molecular imaging.

IMPORTANCE

To understand how spatial and temporal distribution of microbiota impact human health, dynamic tools for monitoring microbiota landscapes inside the host are needed. Particularly when considering the complexity of the gastrointestinal tract and the microbiota that dwell within, tools for monitoring deep segments of the gut non-invasively are required. Medical imaging provides solutions that enable the study of microorganisms in their preferred niche regardless of health status. To bootstrap this technology, we investigated the magnetic resonance imaging (MRI) properties of bacterial isolates and showed that outstanding signal detection is an inherent property of several strains. Among these, we showed that bacteria relying on manganese metabolism have an MRI characteristic that is distinct from mammalian cells. Our findings will lead to direct and safe imaging of bacteria; influence how we monitor both infection and gut health; and help direct the use of antibiotics to curtail the growing threat of antibiotic resistance.
Enterobacteralesplasmid sharing amongst human bloodstream infections, livestock, wastewater, and waterway niches in Oxfordshire, UK
Authors: Matlock William , Lipworth Samuel , Chau Kevin K. , Abu Oun Manal , Barker Leanne , Kavanagh James , Andersson Monique , Oakley Sarah , Morgan Marcus , Crook Derrick W. , Read Daniel S. , Anjum Muna , Shaw Liam P. , Stoesser Nicole ,   REHAB Consortium
Published: May 2022

Plasmids enable the dissemination of antimicrobial resistance (AMR) in commonEnterobacteralespathogens, representing a major public health challenge. However, the extent of plasmid sharing and evolution betweenEnterobacteralescausing human infections and other niches remains unclear, including the emergence of resistance plasmids. Dense, unselected sampling is highly relevant to developing our understanding of plasmid epidemiology and designing appropriate interventions to limit the emergence and dissemination of plasmid-associated AMR. We established a geographically and temporally restricted collection of human bloodstream infection (BSI)-associated, livestock-associated (cattle, pig, poultry, and sheep faeces, farm soils) and wastewater treatment work (WwTW)-associated (influent, effluent, waterways upstream/downstream of effluent outlets)Enterobacterales. Isolates were collected between 2008-2020 from sites <60km apart in Oxfordshire, UK. Pangenome analysis of plasmid clusters revealed shared “backbones”, with phylogenies suggesting an intertwined ecology where well-conserved plasmid backbones carry diverse accessory functions, including AMR genes. Many plasmid “backbones” were seen across species and niches, raising the possibility that plasmid movement between these followed by rapid accessory gene change could be relatively common. Overall, the signature of identical plasmid sharing is likely to be a highly transient one, implying that plasmid movement might be occurring at greater rates than previously estimated, raising a challenge for future genomic One Health studies.

Funding

This study was funded by the Antimicrobial Resistance Cross-council Initiative supported by the seven research councils and the NIHR, UK.
Biophysical characterization of calcium-binding and modulatory-domain dynamics in a pentameric ligand-gated ion channel
Authors: Lycksell Marie , Rovšnik Urška , Hanke Anton , Martel Anne , Howard Rebecca J , Lindahl Erik
Published: May 2022

Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in organisms ranging from bacteria to humans. Among the prokaryotic pLGICs there is architectural diversity involving N-terminal domains (NTDs) not found in eukaryotic relatives, exemplified by the calcium-sensitive channel (DeCLIC) from a Desulfofustis deltaproteobacterium, which has an NTD in addition to the canonical pLGIC structure. Here we have characterized the structure and dynamics of DeCLIC through cryo-electron microscopy (cryo-EM), small-angle neutron scattering (SANS), and molecular dynamics (MD) simulations. In the presence and absence of calcium, cryo-EM yielded structures with alternative conformations of the calcium binding site. SANS profiles further revealed conformational diversity at room temperature beyond that observed in static structures, shown through MD to be largely attributable to rigid body motions of the NTD relative to the protein core, with expanded and asymmetric conformations improving the fit of the SANS data. This work reveals the range of motion available to the DeCLIC NTD and calcium binding site, expanding the conformational landscape of the pLGIC family. Further, these findings demonstrate the power of combining low-resolution scattering, high-resolution structural, and MD-simulation data to elucidate interfacial interactions that are highly conserved in the pLGIC family.
Vertical gradients in photosynthetic physiology diverge at the latitudinal range extremes of white spruce
Authors: Schmiege Stephanie C. , Griffin Kevin L. , Boelman Natalie T. , Vierling Lee A. , Bruner Sarah G. , Min Elizabeth , Maguire Andrew J. , Jensen Johanna , Eitel Jan U. H.
Published: May 2022

Light availability drives vertical canopy gradients in photosynthetic functioning and carbon (C) balance, yet patterns of variability in these gradients remain unclear. We measured light availability, photosynthetic CO2 and light response curves, foliar C, nitrogen (N) and pigment concentrations, and the photochemical reflectance index (PRI) on upper and lower canopy needles of white spruce trees (Picea glauca) at the species’ northern and southern range extremes. We combined our photosynthetic data with previously published respiratory data to compare and contrast canopy C balance between latitudinal extremes. We found steep canopy gradients in irradiance, photosynthesis, and leaf traits at the southern range limit, but a lack of variation across canopy positions at the northern range limit. Thus, unlike many tree species from tropical to mid-latitude forests, high latitude trees may not require vertical gradients of metabolic activity to optimize photosynthetic C gain. Consequently, accounting for self-shading is less critical for predicting gross primary productivity at northern relative to southern latitudes. Northern trees also had a significantly smaller net positive leaf C balance than southern trees suggesting that, regardless of canopy position, low photosynthetic rates coupled with high respiratory costs may ultimately constrain the northern range limit of this widely distributed boreal species.

SUMMARY STATEMENT

Canopy gradients in photosynthetic capacity of white spruce diminish at high compared to low latitudes. Low carbon balance in high latitude trees may determine the extent of northern treeline.
Depth normalization for single-cell genomics count data
Authors: Booeshaghi A. Sina , Hallgrímsdóttir Ingileif B. , Gálvez-Merchán Ángel , Pachter Lior
Published: May 2022

Single-cell genomics analysis requires normalization of feature counts that stabilizes variance while accounting for variable cell sequencing depth. We discuss some of the trade-offs present with current widely used methods, and analyze their performance on 526 single-cell RNA-seq datasets. The results lead us to recommend proportional fitting prior to log transformation followed by an additional proportional fitting.
Crystal structure and biochemical analysis suggest that YjoB ATPase is a substrate-specific molecular chaperone
Authors: Kwon Eunju , Dahal Pawan , Kim Dong Young
Published: May 2022

AAA+ ATPases are ubiquitous proteins associated with most cellular processes, including DNA unwinding and protein unfolding. Their functional and structural properties are typically determined by domains and motifs added to the conserved ATPases domain. Currently, the molecular function and structure of many ATPases remain elusive. Here, we report the crystal structure and biochemical analyses of YjoB, a Bacillus subtilis AAA+ protein. The crystal structure revealed that the YjoB hexamer forms a bucket hat-shaped structure with a porous chamber. Biochemical analyses showed that YjoB prevents the aggregation of vegetative catalase KatA and gluconeogenesis-specific glyceraldehyde-3 phosphate dehydrogenase GapB, but not citrate synthase, a conventional substrate. Structural and biochemical analyses further showed that the internal chamber of YjoB is necessary for the chaperone activity. Our results suggest that YjoB, conserved in the class Bacilli, is a molecular chaperone acting in the starvation/stationary phases of B. subtilis growth.
Mechanism of threonine ADP-ribosylation of F-actin by a Tc toxin
Authors: Belyy Alexander , Lindemann Florian , Roderer Daniel , Funk Johanna , Bardiaux Benjamin , Protze Jonas , Bieling Peter , Oschkinat Hartmut , Raunser Stefan
Published: May 2022

Tc toxins deliver toxic enzymes into host cells by a unique injection mechanism. One of these enzymes is TccC3, an ADP-ribosyltransferase from Photorhabdus luminescens. Once TccC3 is translocated into the target cell, the enzyme ADP-ribosylates actin, resulting in clustering of the actin cytoskeleton and ultimately cell death. Here, we combine biochemistry, solution and solid-state NMR spectroscopy and cryo-EM to show in atomic detail how TccC3 modifies actin. We find that the ADP-ribosyltransferase does not bind to G-actin but interacts with two consecutive actin subunits of F-actin. The binding of TccC3 to F-actin occurs via an induced-fit mechanism that facilitates access of NAD+ to the nucleotide binding pocket. The following nucleophilic substitution reaction results in the transfer of ADP-ribose to threonine-148 of F-actin. We demonstrate that this site-specific modification of F-actin prevents its interaction with depolymerization factors, such as cofilin, which impairs actin network turnover and leads to steady actin polymerization. Our findings reveal in atomic detail a new mechanism of action of a bacterial toxin through specific targeting and modification of F-actin.
Urine-derived exosomes from individuals with IPF carry pro-fibrotic cargo
Authors: Elliot Sharon J. , Catanuto Paola , Pereira-Simon Simone , Xia Xiaomei , Shahzeidi Shahriar , Roberts Evan R. , Ludlow John W. , Hamdan Suzana , Daunert Sylvia , Parra Jennifer , Stone Rivka , Pastar Irena , Tomic-Canic Marjana , Glassberg Marilyn K.
Published: May 2022

Background

MicroRNAs (miRNA) and other components contained in extracellular vesicles may reflect the presence of a disease. Lung tissue, sputum and sera of individuals with idiopathic pulmonary fibrosis (IPF) show alterations in miRNA expression. We designed this study to test whether urine and/or tissue derived exosomal miRNAs from individuals with IPF carry cargo that can promote fibrosis.

Methods

Exosomes were isolated from urine (U-IPFexo), lung tissue myofibroblasts (MF-IPFexo), serum from individuals with IPF (n=16) and age/sex-matched controls without lung disease (n=10). We analyzed microRNA expression of isolated exosomes and their in vivo bio-distribution. We investigated the effect on ex vivo skin wound healing and in in vivo mouse lung models.

Results

U-IPFexo or MF-IPFexo expressed miR let-7d, miR-29a-5p, miR 181b-3p and miR-199a-3p consistent with previous reports of miRNA expression obtained from lung tissue/sera from patients with IPF. In vivo bio-distribution experiments detected bioluminescent exosomes in the lung of normal C57Bl6 mice within 5 minutes after intravenous infusion, followed by distribution to other organs irrespective of exosome source. Exosomes labeled with gold nanoparticles and imaged by transmission electron microscopy were visualized in alveolar epithelial type I and type II cells. Treatment of human and mouse lung punches obtained from control, non-fibrotic lungs with either U-IPFexo or MF-IPFexo produced a fibrotic phenotype. A fibrotic phenotype was also induced in a human ex vivo skin model and in in vivo lung models.

Conclusions

Our results provide evidence of a systemic feature of IPF whereby exosomes contain pro-fibrotic miRNAs when obtained from a fibrotic source and interfere with response to tissue injury as measured in skin and lung models.

Funding

This work was supported in part by Lester and Sue Smith Foundation and The Samrick Family Foundation and NIH grants R21 AG060338 (SE and MKG), U01 DK119085 (IP, RS, MTC).
Transcriptional fluctuations govern the serum dependent cell cycle duration heterogeneities in Mammalian cells
Authors: Govindaraj Vinodhini , Sarma Subrot , Karulkar Atharva , Purwar Rahul , Kar Sandip
Published: May 2022

Mammalian cells exhibit a high degree of intercellular variability in cell cycle period and phase durations. However, the factors orchestrating the cell cycle duration heterogeneities remain unclear. Herein, by combining cell cycle network-based mathematical models with live single-cell imaging studies under varied serum conditions, we demonstrate that fluctuating transcription rates of cell cycle regulatory genes across cell lineages and during cell cycle progression in mammalian cells majorly govern the robust correlation patterns of cell cycle period and phase durations among sister, cousin, and mother-daughter lineage pairs. However, for the overall cellular population, alteration in serum level modulates the fluctuation and correlation patterns of cell cycle period and phase durations in a correlated manner. These heterogeneities at the population level can be finetuned under limited serum conditions by perturbing the cell cycle network using a p38-signaling inhibitor without affecting the robust lineage level correlations. Overall, our approach identifies transcriptional fluctuations as the key controlling factor for the cell cycle duration heterogeneities, and predicts ways to reduce cell-to-cell variabilities by perturbing the cell cycle network regulations.

Significance statement

In malignant tumors, cells display a diverse pattern in cell division time. This cell-to-cell variability in cell cycle duration had been observed even under culture conditions for various mammalian cells. Here we used live-cell imaging studies to monitor FUCCI-HeLa cells and quantified the cell cycle period and time spent in different phases under varied serum conditions. We proposed a set of stochastic cell cycle network-based mathematical models to investigate the live-cell imaging data and unraveled that the transcription rate variation across cell lineages and during cell cycle phases explains every aspect of the cell cycle duration variabilities. Our models identified how different deterministic effects and stochastic fluctuations control these variabilities and predicted ways to alter these cell cycle duration variabilities.
Phasing of de novo mutations using a scaled-up multiple amplicon long-read sequencing approach
Authors: Holt G.S. , Batty L. , Alobaidi B. , Smith H. , Oud M.S. , Ramos L. , Xavier M.J. , Veltman J.A.
Published: May 2022

De novo mutations (DNMs) play an important role in severe genetic disorders that reduce fitness. To better understand the role of DNMs in disease, it is important to determine the parent-of-origin and timing of the mutational events that give rise to the mutations, especially in sex-specific developmental disorders such as male infertility. However, currently available short-read sequencing approaches are not ideally suited for phasing as this requires long continuous DNA strands that span both the DNM and one or more informative SNPs. To overcome these challenges, we optimised and implemented a multiplexed long-read sequencing approach using the Oxford Nanopore technologies MinION platform. We specifically focused on improving target amplification, integrating long-read sequenced data with high-quality short-read sequence data, and developing an anchored phasing computational method. This approach was able to handle the inherent phasing challenges that arise from long-range target amplification and the normal accumulation of sequencing error associated with long-read sequencing. In total, 77 out of 109 DNMs (71%) were successfully phased and parent-of-origin identified. The majority of phased DNMs were prezygotic (90%), the accuracy of which is highlighted by the average mutant allele frequency of 49.6% and a standard error margin of 0.84%. This study demonstrates the benefits of using an integrated short-read and long-read sequencing approach for large-scale DNM phasing.
SimBu: Bias-aware simulation of bulk RNA-seq data with variable cell type composition
Authors: Dietrich Alexander , Sturm Gregor , Merotto Lorenzo , Marini Federico , Finotello Francesca , List Markus
Published: May 2022

Motivation

As complex tissues are typically composed of various cell types, deconvolution tools have been developed to computationally infer their cellular composition from bulk RNA sequencing (RNA-seq) data. To comprehensively assess deconvolution performance, gold-standard datasets are indispensable. Gold-standard, experimental techniques like flow cytometry or immunohistochemistry are resource-intensive and cannot be systematically applied to the numerous cell types and tissues profiled with high-throughput transcriptomics. The simulation of ‘pseudo-bulk’ data, generated by aggregating single-cell RNA-seq (scRNA-seq) expression profiles in pre-defined proportions, offers a scalable and cost-effective alternative. This makes it feasible to create in silico gold standards that allow fine-grained control of cell-type fractions not conceivable in an experimental setup. However, at present, no simulation software for generating pseudo-bulk RNA-seq data exists.

Results

We developed SimBu, an R package capable of simulating pseudo-bulk samples based on various simulation scenarios, designed to test specific features of deconvolution methods. A unique feature of SimBu is the modelling of cell-type-specific mRNA bias using experimentally-derived or data-driven scaling factors. Here, we show that SimBu can generate realistic pseudo-bulk data, recapitulating the biological and statistical features of real RNA-seq data. Finally, we illustrate the impact of mRNA bias on the evaluation of deconvolution tools and provide recommendations for the selection of suitable methods for estimating mRNA content.

Conclusion

SimBu is a user-friendly and flexible tool for simulating realistic pseudo-bulk RNA-seq datasets serving as in silico gold-standard for assessing cell-type deconvolution methods.

Availability

SimBu is freely available at https://github.com/omnideconv/SimBu as an R package under the GPL-3 license.

Contact

alex.dietrich@tum.de and markus.list@tum.de

Supplementary information

Supplementary data are available at Bioinformatics online.
A Saccharomyces cerevisiae Model for Overexpression of Ntg1 a Base Excision DNA Repair Protein Reveals Novel Genetic Interactions
Authors: McPherson Annie J. , Jowhar Ziad M. , Doetsch Paul W. , Corbett Anita H.
Published: May 2022

The Base Excision Repair (BER) pathway repairs oxidative DNA damage, a common and detrimental form of damage to the genome. Although biochemical steps in BER have been well define, little is understood about how the pathway is regulated. Such regulation is critical, as cells must respond rapidly to DNA damage while avoiding aberrant activation of repair proteins that can produce DNA damage as intermediates in the repair pathway. Indeed, overexpression of the human BER protein, NTHL1, a DNA N-glycosylase, can cause genomic instability and early cellular hallmarks of cancer. We developed a Saccharomyces cerevisiae model to explore how overexpression of NTHL1 may impair cellular function. Overexpression of Ntg1, the budding yeast orthologue of NTHL1, impairs cell growth. To dissect mechanisms underlying this growth defect, we overexpressed either wild-type Ntg1 or a catalytically inactive variant of Ntg1 (ntg1catdead). Consistent with results obtained for NTHL1, both variants of Ntg1 impair cell growth, but only the wild-type protein causes accumulation of double-strand breaks and chromosome loss. We screened a panel of DNA repair mutants for resistance/sensitivity to overexpression of wild-type Ntg1 or ntg1catdead. This analysis identified several cellular pathways that protect cells from Ntg1-induced damage, providing insight into interplay between DNA repair pathways. Finally, we identified a link to sumoylation and probed how this post-translational modification could contribute to regulation of Ntg1 function. This study describes a budding yeast system to understand how cells regulate and respond to dysregulation of the BER pathway.

Take Away

Overexpression of a base excision DNA repair protein impairs cell growth

Overexpression of a base excision DNA repair protein can cause DNA damage

Multiple mechanisms cause DNA damage from overexpression of a repair protein

DNA repair pathways functionally interact to protect cells from DNA damage

Precise regulation of the activity of DNA repair proteins is critical
The vectoring competence of the mite Varroa destructor for Deformed wing virus of honey bees is highly dynamic and affects survival of the mite
Authors: Ryabov Eugene V. , Posada-Florez Francisco , Rogers Curtis , Lamas Zachary S. , Evans Jay D. , Chen Yanping , Cook Steven C.
Published: May 2022

The ectoparasitic mite, Varroa destructor and the viruses it vectors, including types A and B of Deformed wing virus (DWV), pose a major threat to honey bees, Apis mellifera. Analysis of 256 mites collected from the same set of colonies on five occasions from May to October 2021 showed that less than a half of them, 39.8% (95% confidence interval (CI): 34.0 - 46.0%), were able to induce an overt-level DWV infection with more than 109 viral genomes per bee in the pupa after 6 days of feeding, with both DWV-A and DWV-B being vectored at similar rates. To investigate the effect of the phoretic stage on the mites’ ability to vector DWV, the mites from two field collection events were divided into two groups, one of which was tested immediately for their infectiveness, and the other was kept with adult worker bees in cages for 12 days prior to testing their infectiveness. We found that while 39.2 % (95% CI: 30.0 – 49.1%) of the immediately tested mites induced overt-level DWV infections, 12-day phoretic passage significantly increased the infectiousness of mites to 89.8% (95% CI: 79.2 – 95.6%). It is likely that Varroa mites that survive brood interruptions in field colonies are increasingly infectious. We found that mite lifespan was significantly affected by the type of DWV it transmitted to pupae. The mites, which induced overt DWV-B but not DWV-A infection had an average lifespan of 15.5 days (95% CI: 11.8 - 19.2 days), which was significantly shorter than those of the mites which induced overt DWV-A but not DWV-B infection, with an average lifespan of 24.3 days (95% CI: 20.2 - 28.5), or the mites which did not induce high levels of DWV-A or DWV-B, with an average survival of 21.2 days (95% CI: 19.0 - 23.5 days). The mites which transmitted high levels of both DWV-A and DWV-B had an intermediate average survival of 20.5 days (95% CI: 15.1 - 25.9 days). The negative impact of DWV-B on mite survival could be a consequence of the ability of DWV-B, but not DWV-A to replicate in Varroa mites.
Spatio-temporal diversity of dietary preferences and stress sensibilities of early and middle Miocene Rhinocerotidae from Eurasia: Impact of climate changes
Authors: Hullot Manon , Merceron Gildas , Antoine Pierre-Olivier
Published: May 2022

Major climatic and ecological changes are documented in terrestrial ecosystems during the Miocene epoch. The Rhinocerotidae are a very interesting clade to investigate the impact of these changes on ecology, as they are abundant and diverse in the fossil record throughout the Miocene. Here, we explored the spatio-temporal evolution of rhinocerotids’ paleoecology during the early and middle Miocene of Europe and Pakistan. We studied the dental texture microwear (proxy for diet) and enamel hypoplasia (stress indicator) of 19 species belonging to four sub-tribes and an unnamed clade of Rhinocerotidae, and coming from nine Eurasian localities ranging from Mammal Neogene zone (MN) 2 to MN7/8. Our results suggest clear differences in the feeding ecology and thus niche partitioning at Kumbi 4 (MN2, Pakistan), Sansan (MN6, France), and Villefranche d’Astarac (MN7/8, France), while overlap of the interpreted diets and subtle variations are discussed for Béon 1 (MN4, France) and Gračanica (MN5/6, Bosnia-Herzegovina). All rhinocerotids studied were interpreted as browsers or mixed-feeders, and none had a grazer nor frugivore diet. The prevalence of hypoplasia was moderate (∼10%) to high (> 20%) at all localities but Kumbi 4 (∼6%), and documented quite well the local conditions. For instance, the high prevalence at the close to Miocene Climatic Optimum locality of Béon 1 (∼26%) has been correlated with periodical droughts, while the moderate ones (∼10%) at Sansan and Devínska Nová Ves Spalte (Slovakia) both dated from the MN6 (i.e., by the middle Miocene Climatic Transition, ca. 13.9 Mya) were linked to the persistence of sub-tropical local conditions. Besides locality, species and tooth locus were also important factors of variation for the prevalence of hypoplasia. The very large hippo-likeBrachypotherium brachypuswas one of the most affected species at all concerned localities (but Sansan), whereas early-diverging elasmotheriines were very little affected, suggesting an influence of phylogeny and/or diet in stress susceptibility.
Subthalamic beta bursts correlate with dopamine-dependent motor symptoms in 106 Parkinson’s patients
Authors: Lofredi Roxanne , Okudzhava Liana , Irmen Friederike , Brücke Christof , Huebl Julius , Krauss Joachim K. , Schneider Gerd-Helge , Faust Katharina , Neumann Wolf-Julian , Kühn Andrea A.
Published: May 2022

Pathologically increased beta power has been described as a biomarker for Parkinson’s disease (PD) and related to prolonged bursts of subthalamic beta synchronization. Here, we investigate the association between subthalamic beta dynamics and motor impairment in a cohort of 106 Parkinson’s patients in the ON- and OFF-medication state, suing two different methods of beta burst determination. We report a frequency-specific correlation of low beta power and burst duration with motor impairment OFF dopaminergic medication. Furthermore, reduction of power and burst duration correlated significantly with symptom alleviation through dopaminergic medication. Importantly, qualitatively similar results were yielded with two different methods of beta burst definition. Our findings validate the robustness of previous results on pathological changes in subcortical oscillations both in the frequency-as well as in the time-domain in the largest cohort of PD patients to date with important implications for next-generation adaptive deep brain stimulation control algorithms.