bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of Whole-Person Eigen-Polygenic Risk Scores with Alzheimer’s Disease
Authors: Kharaghani Amin , Tio Earvin , Milic Milos , Bennett David A. , De Jager Philip L. , Schneider Julie A. , Sun Lei , Felsky Daniel
Published: September 2022

Late-Onset Alzheimer’s Disease (LOAD) is a heterogeneous neurodegenerative disorder with complex etiology and high heritability. Its multifactorial risk profile and large portions of unexplained heritability suggest the involvement of yet unidentified genetic risk factors. Here we describe the “whole person” genetic risk landscape of polygenic risk scores for 2,218 traits in 2,044 elderly individuals and test if novel eigen-PRSs derived from clustered subnetworks of single-trait PRSs can improve prediction of LOAD diagnosis, rates of cognitive decline, and canonical LOAD neuropathology. Principal component analyses of thousands of PRSs found generally poor global correlation among traits. However, component loadings confirmed covariance of clinically and biologically related traits and diagnoses, with the top PCs representing autoimmune traits, cardiovascular traits, and general pain medication prescriptions, depending on the PRS variant inclusion threshold. Network analyses revealed distinct clusters of PRSs with clinical and biological interpretability. Novel eigen-PRSs (ePRS) derived from these clusters were significantly associated with LOAD-related phenotypes and improved predictive model performance over the state-of-the-art LOAD PRS alone. Notably, an ePRS representing clusters of traits related to cholesterol levels was able to improve variance explained in a model of brain-wide beta-amyloid burden by 1.7% (likelihood ratio test p=9.02×10−7). While many associations of ePRS with LOAD phenotypes were eliminated by the removal of APOE-proximal loci, some modules (e.g. retinal defects, acidosis, colon health, ischaemic heart disease) showed associations at an unadjusted type I error rate. Our approach reveals new relationships between genetic risk for vascular, inflammatory, and other age-related traits and offers improvements over the existing single-trait PRS approach to capturing heritable risk for cognitive decline and beta-amyloid accumulation. Our results are catalogued for the scientific community, to aid in the generation of new hypotheses based on our maps of clustered PRSs and associations with LOAD-related phenotypes.
Diacammaants adjust liquid foraging strategies in response to biophysical constraints
Authors: Fujioka Haruna , Marchand Manon , LeBoeuf Adria C.
Published: September 2022

Ant foragers need to provide food to the rest of the colony, which often requires transport over long distances. Foraging for liquid is especially challenging because it is difficult to transport and share. Many social insects store liquid food inside the crop to transport it to the nest, and then regurgitate this fluid to distribute it to nestmates through a behaviour called trophallaxis. Some ants instead transport fluids with a riskier behaviour called pseudotrophallaxis – holding a drop of liquid between the mandibles through surface tension. Ants can then share this droplet with nestmates without ingestion or regurgitation. Here, we hypothesised that ants optimise their liquid-collection approach depending on viscosity. Working with a ponerine ant that uses both trophallaxis and pseudotrophallaxis, we investigated why each liquid-collection behaviour might be favoured under different conditions by measuring biophysical properties, collection time, and reaction to food quality for typical and viscosity-altered sucrose solutions. We found that ants can collect more liquid food per unit time by mandibular grabbing than by drinking. At high viscosities, which in nature correspond to high sugar concentrations, ants switched their liquid collection method to mandibular grabbing in response to viscosity, and not to sweetness. Our results demonstrate that ants change their transport and sharing methods according to viscosity – a proxy for sugar concentration in nature – increasing the mass of sugar returned to the nest per trip.
Identification of interpretable clusters and associated signatures in breast cancer single cell data: a topic modeling approach
Authors: Gabriele Malagoli , Filippo Valle , Emmanuel Barillot , Michele Caselle , Loredana Martignetti
Published: September 2022

Topic modeling is a popular technique in machine learning and natural language processing, where a corpus of text documents is classified into themes or topics using word frequency analysis. This approach has proven successful in various biological data analysis applications, such as predicting cancer subtypes with high accuracy and identifying genes, enhancers, and stable cell types simultaneously from sparse single cell epigenomics data. The advantage of using a topic model is that it not only serves as a clustering algorithm, but it can also explain clustering results by providing word probability distributions over topics.

Our study proposes a novel topic modeling approach for clustering single cells and detecting topics (gene signatures) in single-cell datasets that measure multiple omics simultaneously. We applied this approach to examine the transcriptional heterogeneity of luminal and triple negative breast cancer cells using patient-derived xenograft models with acquired resistance to chemotherapy and targeted therapy. Through this approach, we identified protein-coding genes and long noncoding RNAs (lncRNAs) that group thousands of cells into biologically similar clusters, accurately distinguishing drug-sensitive and resistant breast cancer types. In comparison to standard state-of-the-art clustering analyses, our approach offers optimal partitioning of genes into topics and cells into clusters simultaneously, producing easily interpretable clustering outcomes. Additionally, we demonstrate that an integrative clustering approach, which combines the information from mRNAs and lncRNAs treated as disjoint omics layers, enhances the accuracy of cell classification.
Submicron-sized in-situ osmotic pressure sensors for in-vitro applications in biology
Authors: Zhang Wenbo , Bertinetti Luca , Yavuzsoy Efe Cuma , Gao Changyou , Schneck Emanuel , Fratzl Peter
Published: September 2022

Physical forces are important cues in determining the development and the normal function of biological tissues. While forces generated by molecular motors have been widely studied, forces resulting from osmotic gradients have been less considered in this context. A possible reason is the lack of direct in-situ measurement methods that can be applied to cell and organ culture systems. Herein, novel kinds of FRET (resonance energy transfer)-based liposomal sensors are developed, so that their sensing range and sensitivity can be adjusted to satisfy physiological osmotic conditions. Several types of sensors are prepared, either based on PEGylated liposomes with steric stabilization and stealth property or on crosslinked liposomes capable of enduring relatively harsh environments for liposomes (e.g. in the presence of biosurfactants). The sensors are demonstrated to be effective in the measurement of osmotic pressures in pre-osteoblastic in-vitro cell culture systems by means of FRET microscopy. This development paves the way towards the in-situ sensing of osmotic pressures in biological culture systems.
Derepression masquerades as activation in a pentameric ligand-gated ion channel
Authors: Tessier Christian J.G. , Emlaw Johnathon R. , Sturgeon Raymond M. , daCosta Corrie J.B.
Published: September 2022

Agonists are ligands that bind to receptors and activate them. In the case of ligand-gated ion channels, such as the muscle-type nicotinic acetylcholine receptor, mechanisms of agonist activation have been studied for decades. Taking advantage of a reconstructed ancestral muscle-type β-subunit that forms spontaneously activating homopentamers, here we show that incorporation of human muscle-type α-subunits represses spontaneous activity, and furthermore that the presence of agonist relieves this α-subunit-dependent repression. Our results demonstrate that rather than provoking channel activation/opening, agonists may instead ‘inhibit the inhibition’ of intrinsic spontaneous activity. Thus, agonist activation may be the apparent manifestation of agonist-induced derepression. These results provide insight into intermediate states that precede channel opening and have implications for the interpretation of agonism in ligand-gated ion channels.
Dissecting the Thermodynamics of ATP Binding to GroEL One Nucleotide at a Time
Authors: Walker Thomas , Sun He Mirabel , Gunnels Tiffany , Wysocki Vicki , Laganowsky Arthur , Rye Hays , Russell David
Published: September 2022

Understanding how large, oligomeric protein complexes respond to the binding of small ligands and other proteins is essential for describing the molecular basis of life. This in turn requires a complete characterization of the binding energetics and correlation of thermodynamic data with interacting structures, including effects of small molecules and solvent. However, the size of many protein oligomers, the myriad intermediate ligation states they can populate, and their often complex allosteric regulation typically restrict analysis by traditional methods to low resolution, ensemble averages. Here, we employ variabletemperature electrospray ionization native mass spectrometry to determine the thermodynamics for stepwise binding of up to ATP molecules to the 801 kDa GroEL complex, a tetradecamer chaperonin complex. Binding thermodynamics reveal strong enthalpy-entropy compensation (EEC) and high degrees of cooperativity are observed for formation of GroEL-ATP7and GroEL-ATP14. These are evidenced by entropically favored ATP binding to thecisring (formation of GroEL-ATP1-7), with variations in EEC for subsequent binding of ATP to thetransring (GroEL-ATP8-14), as expected for negative inter-ring cooperativity. Entropy driven ATP binding to the GroEL tetradecamer is consistent with ligand induced conformational changes of the GroEL tetradecamer, though the magnitude of the entropy change suggests that reorganization of GroEL-hydrating water molecules and/or expulsion of water from the GroEL cavity may also play a key role. By determining the thermodynamic signatures for individual ligand binding reactions to the large, nearly MDa GroEL complex, we expand our fundamental view of chaperonin functional chemistry. Moreover, this work and related studies of protein-ligand interactions illustrate unparalleled capabilities of vT-ESI-nMS for thermodynamics studies of protein interactions with ligands, and other molecules, such as proteins and drugs.
The intrinsically disordered protein glue of myelin: Linking AlphaFold2 predictions to experimental data
Authors: Krokengen Oda C. , Raasakka Arne , Kursula Petri
Published: September 2022

Numerous human proteins are either partially or fully classified as intrinsically disordered proteins (IDPs). Due to their properties, high-resolution structural information about IDPs is generally lacking. On the other hand, IDPs are known to adopt local ordered structures upon interactions with ligands, which could bee.g. other proteins or lipid membrane surfaces. While recent developments in protein structure prediction have been revolutionary, their impact on IDP research at high resolution remains limited. We took a specific example of two myelin-specific IDPs, the myelin basic protein (MBP) and the cytoplasmic domain of myelin protein zero (P0ct). Both of these IDPs are known to be crucial for normal nervous system development and function, and while they are disordered in solution, upon membrane binding, they partially fold into helices, being embedded into the lipid membrane. We carried out AlphaFold2 predictions of both proteins and analysed the models in light of previously published data related to solution structure and molecular interactions. We observe that the predicted models have helical segments that closely correspond to the characterised membrane-binding sites on both proteins. We furthermore analyse the fits of the models to SAXS data from the same IDPs. Artificial intelligence-based models of IDPs appear to be able to provide detailed information on the ligand-bound state of these proteins, instead of the form dominating free in solution. We further discuss the implications of the predictions for normal mammalian nervous system myelination and their relevance to understanding disease aspects of these IDPs.
Convergent community assembly among globally separated acidic cave biofilms
Authors: Jones Daniel , Schaperdoth Irene , Northup Diana E. , Gómez-Cruz Rodolfo , Macalady Jennifer L.
Published: September 2022

Acidophilic bacteria and archaea inhabit extreme geochemical ‘islands’ that can tell us when and how geographic barriers affect the biogeography of microorganisms. Here we describe microbial communities from extremely acidic (pH 0-1) biofilms known as “snottites” from hydrogen sulfide-rich caves around the world. Given the extreme acidity and subsurface location of these biofilms, and in light of earlier work showing strong geographic patterns among snottite Acidithiobacillus populations, we investigated their structure and diversity in order to understand how geography might impact community assembly. We used 16S rRNA gene cloning and fluorescence in situ hybridization (FISH) to investigate 26 snottite samples from four sulfidic caves in Italy and Mexico. All samples had very low biodiversity and were dominated by sulfur-oxidizing bacteria in the genus Acidithiobacillus. Ferroplasma and other archaea in the Thermoplasmatales ranged from 0 to 50% of total cells, and relatives of the bacterial genera Acidimicrobium and Ferrimicrobium were up to 15% of total cells. Rare phylotypes included Sulfobacillus spp. and members of the Dependentiae and Saccharibacteria (formerly TM6 and TM7). Although the same genera of acidophiles occurred in snottites on separate continents, most members of those genera represent substantially divergent populations with 16S rRNA genes that are only 95-98% similar. Our findings are consistent with a model of community assembly where sulfidic caves are stochastically colonized by microorganisms from local sources, which are strongly filtered through selection for extreme acid tolerance, and these different colonization histories are maintained by dispersal restrictions within and among caves.

Importance

Microorganisms that are adapted to extremely acidic conditions, known as extreme acidophiles, are catalysts for rock weathering, metal cycling, and mineral formation in naturally acidic environments. They are also important drivers of large-scale industrial processes such as biomining and contaminant remediation. Understanding the factors that govern their ecology and distribution can help us better predict and utilize their activities in natural and engineered systems. However, extremely acidic habitats are unusual in that they are almost always isolated within circumneutral landscapes. So where did their acid-adapted inhabitants come from, and how do new colonists arrive and become established? In this study, we took advantage of a unique natural experiment in Earth’s subsurface to show how isolation may have played a role in the colonization history, community assembly, and diversity of highly acidic microbial biofilms.
Validation and Establishment of a SARS-CoV-2 Lentivirus Surrogate Neutralization Assay as a pre-screening tool for the Plaque Reduction Neutralization Test
Authors: Merluza John , Ung Johnny , Makowski Kai , Robinson Alyssia , Manguiat Kathy , Mueller Nicole , Audet Jonathan , Chen Julie Chih-Yu , Strong James E , Wood Heidi , Bello Alexander
Published: September 2022

Neutralization assays are important in understanding and quantifying neutralizing antibody responses towards SARS-CoV-2. The SARS-CoV-2 Lentivirus Surrogate Neutralization Assay (SCLSNA) can be used in biosafety level 2 (BSL-2) laboratories and has been shown to be a reliable, alternative approach to the plaque reduction neutralization test (PRNT). In this study, we optimized and validated the SCLSNA to assess its ability as a comparator and pre-screening method to support the PRNT. Comparability between the PRNT and SCLSNA was determined through clinical sensitivity and specificity evaluations. Clinical sensitivity and specificity produced acceptable results with 100% (95% CI: 94-100) specificity and 100% (95% CI: 94-100) sensitivity against ancestral Wuhan spike pseudotyped lentivirus. The sensitivity and specificity against B.1.1.7 spike pseudotyped lentivirus resulted in 88.3% (95% CI: 77.8 to 94.2) and 100% (95% CI: 94-100), respectively. Assay precision measuring intra-assay variability produced acceptable results for High (1:≥ 640 PRNT50), Mid (1:160 PRNT50) and Low (1:40 PRNT50) antibody titer concentration ranges based on the PRNT50, with %CV of 14.21, 12.47, and 13.28 respectively. Intermediate precision indicated acceptable ranges for the High and Mid concentrations, with %CV of 15.52 and 16.09, respectively. However, the Low concentration did not meet the acceptance criteria with a %CV of 26.42. Acceptable ranges were found in the robustness evaluation for both intra-assay and inter-assay variability. In summary, the validation parameters tested met the acceptance criteria, making the SCLSNA method fit for its intended purpose, which can be used to support the PRNT.
Antibodies against the flotillin-1/2 complex in patients with multiple sclerosis
Authors: Lleixà Cinta , Caballero-Ávila Marta , Pascual-Goñi Elba , Martín-Aguilar Lorena , Vidal Nuria , Tejada Clara , Valdés-Hevia Eduardo , Zárate Elisa , Vesperinas Ana , Collet Roger , Franco Teresa , Martínez-Martínez Laura , Cortés-Vicente Elena , Rojas-García Ricard , Gómez-Anson Beatriz , Gil Anna , González Cristina , Brieva Luis , Martínez-Yélamos Sergio , Querol Luis
Published: September 2022

Multiple sclerosis (MS) is a tissue-specific autoimmune disease of the central nervous system in which the antigen(s) remains elusive. Antibodies targeting the flotillin-1/2 (FLOT–1/2) complex have been described in 1-2% of the patients in a recent study. Other candidate antigens as anoctamin-2 (ANO2) or neurofascin-155 (NF155) have been previously described in MS patients, although their clinical relevance remains uncertain. Our study aims to analyse the frequency and clinical relevance of antibodies against NF155, ANO2 and the FLOT-1/2 complex in MS.

Serum (n=252) and CSF (n=50) samples from 282 MS patients were included in the study. The control group was composed of 260 serum samples (71 healthy donors and 189 with other neuroinflammatory disorders). Anti-FLOT1/2, anti-ANO2 and anti-NF155 antibodies were tested by cell-based assays using transfected-HEK293 cells. We identified 6 MS patients with antibodies against the FLOT-1/2 complex (2.1%) and 1 MS patient with antibodies against ANO2 (0.35%). All MS patients were negative for anti-NF155 antibodies. Three of the anti-FLOT1/2 positive patients showed anti-FLOT-1/2 positivity in other serum samples extracted at different moments of their disease. IgG subclasses of anti-FLOT-1/2 antibodies were predominantly IgG1 and IgG3.

We confirm that antibodies targeting the Flotillin-1/2 complex are present in a subgroup of patients with MS. Further studies are needed to understand the clinical and pathological relevance of anti-FLOT-1/2 autoantibodies in MS.
The SweDen risk score- predicting death 1-year after myocardial infarction
Authors: Rylance Rebecca T. , Wagner Philippe , Olesen Kevin K. W. , Carlson Jonas , Alfredsson Joakim , Jernberg Tomas , Leosdottir Margret , Johansson Pelle , Vasko Peter , Maeng Michael , Mohammad Moman A. , Erlinge David
Published: September 2022

Objectives

Our aim was to derive, based on the SWEDEHEART registry, and validate, using the Western Denmark Heart registry, a patient-oriented risk score, the SweDen score, which could calculate the risk of 1-year mortality following a myocardial infarction (MI).

Methods

The factors included in the SweDen score were age, sex, smoking, diabetes, heart failure, and statin use. These were chosen a priori by the SWEDEHEART steering group based on the premise that the factors were information known by the patients themselves. The score was evaluated using various statistical methods such as time-dependent receiver operating characteristics curves of the linear predictor, area under the curve metrics, Kaplan-Meier survivor curves, and the calibration slope.

Results

The area under the curve values were 0.81 in the derivation data and 0.76 in the validation data. The Kaplan-Meier curves showed similar patient profiles across datasets. The calibration slope was 1.03 (95% CI 0.99-1.08) in the validation data using the linear predictor from the derivation data.

Conclusions

The SweDen risk score is a novel tool created for patient use. The risk score calculator will be available online and presents mortality risk on a colour scale to simplify interpretation and to avoid exact life span expectancies. It provides a validated patient-oriented risk score predicting the risk of death at 1 year after suffering a MI, which visualizes the benefit of statin use and smoking cessation in a simple way.
Identification of tubulin polymerization inhibitors with a CRISPR-edited cell line with endogenous fluorescent tagging of β-tubulin and Histone 1
Authors: Khachatryan Harutyun , Barrero Carlos A. , Gordon John , Olszowy Bartlomiej , Perez-Leal Oscar
Published: September 2022

Tubulin is an essential protein to maintain the cellular structure and for the cell division process. Inhibiting tubulin polymerization has proven to be an effective method for slowing cancer cell growth. Traditionally, identifying tubulin polymerization inhibitors involved using pure tubulin for in vitro assays or procedures using cells that require cell fixing and anti-tubulin antibody staining. This study explores using a cell line developed via CRISPR genome editing as a cell model to identify tubulin polymerization inhibitors with live cells without using exogenous staining. The cell line has endogenous tagging with fluorescent proteins of β-tubulin and a nuclear protein to facilitate image cellular segmentation by high-content imaging analysis (HCI). The cells were treated with known tubulin polymerization inhibitors, colchicine and vincristine, and the presence of phenotypic changes that indicate tubulin polymerization inhibition were confirmed via HCI. A library of 429 kinase inhibitors was screened to discover tubulin polymerization inhibitors and three compounds that inhibit tubulin polymerization were found (ON-01910, HMN-214, and KX2-391). Live cell tracking analysis confirms that depolymerization of tubulin occurs rapidly after compound treatments. These results suggest that CRISPR-edited cells with fluorescent endogenous tagging of β-tubulin can be used to screen larger compound libraries containing diverse chemical families to identify novel tubulin polymerization inhibitors.
Deficits in cerebellum-dependent learning and cerebellar morphology in male and female BTBR autism model mice
Authors: Kiffmeyer Elizabeth A. , Cosgrove Jameson A. , Siganos Jenna K. , Bien Heidi E. , Vipond Jade E. , Vogt Karisa R. , Kloth Alexander D.
Published: September 2022

Recently, there has been increased interest in the role of the cerebellum in autism spectrum disorders (ASD). In order to better understand the pathophysiological role of the cerebellum in ASD, it is necessary to have a variety of mouse models that have face validity for cerebellar disruption in humans. Here, we add to the literature on the cerebellum transgenic and induced mouse models of autism with the characterization of the cerebellum in the BTBR T+Itpr3tf/J (BTBR) inbred mouse strain, which has behavioral phenotypes that are suggestive of ASD in patients. When we examined both male and female adult BTBR mice in comparison to C57BL/6J (C57) controls, we noted that both mice showed motor coordination deficits characteristic of cerebellar function, but only the male mice showed differences in delay eyeblink conditioning, a cerebellum-dependent learning task that is also disrupted in ASD patients. Both male and female BTBR mice showed considerable expansion of and abnormal foliation in the cerebellum vermis--including significant expansion of specific lobules in the anterior cerebellum. In addition, we found a slight but significant decrease in Purkinje cell density in both male and female BTBR mice, irrespective of lobule. Furthermore, there was a marked reduction of Purkinje cell dendritic spines density in both male and female BTBR mice. These findings suggest that, for the most part, the BTBR mouse model successfully phenocopies many of the characteristics of the subpopulation of ASD patients that have a hypertrophic cerebellum. We discuss the significance of sex differences--revealed for the first time in BTBR mice, and present in only a small number of cerebellum studies--and the importance of concordance on other metrics between male and female BTBR mice.

Graphical Abstract

Summary of differences between BTBR mice (left) and C57 mice (right) demonstrated by this study, separated by sex.
Central metabolism is a key player in E. coli biofilm stimulation by sub-MIC antibiotics
Authors: Yaeger Luke N. , French Shawn , Brown Eric D. , Côté Jean Philippe , Burrows Lori L.
Published: September 2022

Exposure of Escherichia coli to sub-inhibitory antibiotics stimulates biofilm formation through poorly characterized mechanisms. Using a high-throughput Congo Red binding assay to report on biofilm matrix production, we screened ∼4000 E. coli K12 deletion mutants for deficiencies in this biofilm stimulation response. Mutants lacking acnA, nuoE, or lpdA failed to respond to sub-MIC novobiocin, implicating central metabolism and aerobic respiration in biofilm stimulation. These genes are members of the ArcA/B regulon – controlled by a respiration-sensitive two-component system. Mutants of arcA and arcB had a ‘pre-activated’ phenotype, where biofilm formation was already high relative to wild type in vehicle control conditions and failed to increase further with the addition of sub-MIC antibiotics. Supporting a role for respiratory stress, the biofilm stimulation response was inhibited when nitrate was provided as an alternative electron acceptor. Deletion of genes encoding the nitrate respiratory machinery abolished its effects, and nitrate respiration increased during growth with sub-MIC antibiotics. In probing the generalizability of biofilm stimulation, we found that the stimulation response to translation inhibitors was minimally affected by nitrate supplementation. Finally, using a metabolism-sensitive dye, we showed spatial co-localization of increased respiration with sub-MIC bactericidal antibiotics. By characterizing the biofilm stimulation response to sub-MIC antibiotics at a systems level, we identified multiple avenues for design of therapeutics that impair bacterial stress management.
Novel lipid biomarkers for algal resistance to viral infection in the ocean
Authors: Schleyer Guy , Kuhlisch Constanze , Ziv Carmit , Ben-Dor Shifra , Malitsky Sergey , Schatz Daniella , Vardi Assaf
Published: September 2022

Marine viruses play a key role in regulating phytoplankton populations, greatly affecting the biogeochemical cycling of major nutrients in the ocean. Resistance to viral infection has been reported for various phytoplankton species under laboratory conditions. Nevertheless, the occurrence of resistant cells in natural populations is underexplored due to the lack of sensitive tools to detect these rare phenotypes. Consequently, our current understanding of the ecological importance of resistance and its underlying mechanisms is limited. Here, we sought to discover lipid biomarkers for the resistance of the bloom-forming alga Emiliania huxleyi to its specific virus, E. huxleyi virus (EhV). We identified novel glycosphingolipids (GSLs) that characterize resistant E. huxleyi strains by applying an untargeted lipidomics approach. Further, we detected these lipid biomarkers in E. huxleyi isolates that were recently collected from E. huxleyi blooms and used them to detect resistant cells in the demise phase of an open ocean E. huxleyi bloom. Lastly, we show that the GSL composition of E. huxleyi cultures that recover following infection and gain resistance to the virus resembles that of resistant strains. These findings highlight the metabolic plasticity and co-evolution of the GSL biosynthetic pathway and underscore its central part in this host-virus arms race.
The SARS-CoV-2 spike N-terminal domain engages 9-O-acetylated α2-8-linked sialic acids
Authors: Tomris Ilhan , Unione Luca , Nguyen Linh , Zaree Pouya , Bouwman Kim M. , Liu Lin , Li Zeshi , Fok Jelle A. , Ríos Carrasco María , van der Woude Roosmarijn , Kimpel Anne L.M. , Linthorst Mirte W. , Verpalen Enrico C.J.M , Caniels Tom G. , Sanders Rogier W. , Heesters Balthasar A. , Pieters Roland J. , Jiménez-Barbero Jesús , Klassen John S. , Boons Geert-Jan , de Vries Robert P.
Published: September 2022

SARS-CoV-2 viruses engage ACE2 as a functional receptor with their spike protein. The S1 domain of the spike protein contains a C-terminal receptor-binding domain (RBD) and an N-terminal domain (NTD). The NTD of other coronaviruses includes a glycan-binding cleft. However, for the SARS-CoV-2 NTD protein-glycan binding was only observed weakly for sialic acids with highly sensitive methods. Amino acid changes in the NTD of Variants of Concern (VoC) shows antigenic pressure, which can be an indication of NTD-mediated receptor binding. Trimeric NTD proteins of SARS-CoV-2, Alpha, Beta, Delta, and Omicron did not reveal a receptor binding capability. Unexpectedly, the SARS-CoV-2 Beta subvariant strain (501Y.V2-1) NTD binding to Vero E6 cells was sensitive to sialidase pretreatment. Glycan microarray analyses identified a putative 9-O-acetylated sialic acid as a ligand, which was confirmed by catch-and-release ESI-MS, STD-NMR analyses, and a graphene-based electrochemical sensor. The Beta (501Y.V2-1) variant attained an enhanced glycan binding modality in the NTD with specificity towards 9-O-acetylated structures, suggesting a dual-receptor functionality of the SARS-CoV-2 S1 domain, which was quickly selected against. These results indicate that SARS-CoV-2 can probe additional evolutionary space, allowing binding to glycan receptors on the surface of target cells.

Graphical abstract

Synopsis

Coronaviruses utilize their N-terminal domain (NTD) for initial reversible low-affinity interaction to (sialylated) glycans. This initial low-affinity/high-avidity engagement enables viral surfing on the target membrane, potentially followed by a stronger secondary receptor interaction. Several coronaviruses, such as HKU1 and OC43, possess a hemagglutinin-esterase for viral release after sialic acid interaction, thus allowing viral dissemination. Other coronaviruses, such as MERS-CoV, do not possess a hemagglutinin-esterase, but interact reversibly to sialic acids allowing for viral surfing and dissemination. The early 501Y.V2-1 subvariant of the Beta SARS-CoV-2 Variant of Concern has attained a receptor-binding functionality towards 9-O-acetylated sialic acid using its NTD. This binding functionality was selected against rapidly, most likely due to poor dissemination. Ablation of sialic acid binding in more recent SARS-CoV-2 Variants of Concern suggests a fine balance of sialic acid interaction of SARS-CoV-2 is required for infection and/or transmission.
Molecular elucidation of drug-induced abnormal assemblies of the Hepatitis B Virus capsid protein by solid-state NMR
Authors: Lecoq Lauriane , Brigandat Louis , Huber Rebecca , Fogeron Marie-Laure , Callon Morgane , Malär Alexander , Wang Shishan , Dujardin Marie , Briday Mathilde , Wiegand Thomas , Durantel David , Burdette Dara , Berke Jan Martin , Meier Beat H. , Nassal Michael , Böckmann Anja
Published: September 2022

Hepatitis B virus (HBV) capsid assembly modulators (CAMs) represent a new class of anti-HBV antivirals. CAMs disturb proper nucleocapsid assembly, by inducing formation of either aberrant assemblies (CAM-A) or of apparently normal but genome-less empty capsids (CAM-E). Classical structural approaches have revealed the CAM binding sites on the capsid protein (Cp), but conformational information on the CAM-induced off-path aberrant assemblies is lacking. We show that solid-state NMR can provide such information, including for wild-type full-length Cp183, and we find that in these assemblies, the asymmetric unit comprises a single Cp molecule rather than the four quasi-equivalent conformers typical for the icosahedral T=4 symmetry of the normal HBV capsids. Furthermore, while in contrast to truncated Cp149, full-length Cp183 assemblies appear, on the mesoscopic level, unaffected by CAM-A, NMR reveals that on the molecular level, Cp183 assemblies are equally aberrant. Finally, we use a eukaryotic cell-free system to reveal how CAMs modulate capsid-RNA interactions and capsid phosphorylation. Our results establish a structural view on assembly modulation of the HBV capsid, and they provide a rationale for recently observed differences between in-cell versus in vitro capsid assembly modulation.
pWCP is a widely distributed and highly conserved Wolbachia plasmid in Culex mosquitoes worldwide
Authors: Ghousein Amani , Tutagata Jordan , Etienne Manuel , Chaumeau Victor , Boyer Sebastien , Pages Nonito , Roiz David , Eren A. Murat , Cambray Guillaume , Reveillaud Julie
Published: September 2022

Mosquitoes represent the most important pathogen vectors and are responsible for the spread of a wide variety of poorly treatable diseases. Wolbachia are obligate intracellular bacteria that are widely distributed among arthropods and collectively represents one of the most promising solutions for vector control. In particular, Wolbachia has been shown to limit the transmission of pathogens, and to dramatically affect the reproductive behavior of their host through its phage WO. While much research has focused on deciphering and exploring the biocontrol applications of these WO-related phenotypes, the extent and potential impact of the Wolbachia mobilome remain poorly appreciated. Notably, several Wolbachia plasmids, carrying WO-like genes and Insertion Sequences (IS), thus possibly interrelated to other genetic units of the endosymbiont, have been recently discovered. Here we investigated the diversity and biogeography of the first described plasmid of Wolbachia in Culex pipiens (pWCP) in several islands and continental countries around the world—including Cambodia, Guadeloupe, Martinique, Thailand, and Mexico—together with mosquito strains from colonies that evolved for 2 to 30 years in the laboratory. Together with earlier observation, our results show that pWCP is omnipresent and strikingly conserved among Wolbachia populations within mosquitoes from distant geographies and environmental conditions. These data suggest a critical role for the plasmid in Wolbachia ecology and evolution, and the potential of a great tool for the further genetic dissection or potential manipulation of the endosymbiont.
Capillary electrophoresis mass spectrometry identifies new isomers of inositol pyrophosphates in mammalian tissues
Authors: Qiu Danye , Gu Chunfang , Liu Guizhen , Ritter Kevin , Eisenbeis Verena B. , Bittner Tamara , Gruzdev Artiom , Seidel Lea , Bengsch Bertram , Shears Stephen B. , Jessen Henning J.
Published: September 2022

Technical challenges have to date prevented a complete profiling of the levels of myo-inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) in mammalian tissues. Here, we have deployed capillary electrophoresis mass spectrometry to identify and record the levels of InsPs and PP-InsPs in several tissues obtained from wild type mice and a newly-created PPIP5K2 knockout strain. We observe that the mouse colon harbours unusually high levels of InsPs and PP-InsPs. Additionally, the PP-InsP profile is considerably more complex than previously reported for animal cells: using chemically synthesized internal stable isotope references, and high-resolution mass spectra, we characterize two new PP-InsP isomers as 4/6-PP-InsP5and 2-PP-InsP5. The latter has not previously been described in Nature. Analysis of feces and the commercial mouse diet suggest the latter is one potential source of noncanonical isomers in the colon. However, we also identify both molecules in the heart, indicating unknown synthesis pathways in mammals. We also demonstrate that the CE-MS method is sensitive enough to measure PP-InsPs from patient samples such as colon biopsies and peripheral blood mononuclear cells (PBMCs). Strikingly, PBMCs also contain 4/6-PP-InsP5and 2-PP-InsP5. In summary, our study substantially expands PP-InsP biology in mammals.
Cryo-EM reveals the membrane binding phenomenon of EspB, a virulence factor of the Mycobacterial Type VII secretion system
Authors: Sengupta Nayanika , P. Surekha , Dutta Somnath
Published: September 2022

Mycobacterium tuberculosis utilizes sophisticated machinery called the type VII secretion system to translocate virulence factors across its complex lipid membrane. ESX-1 is one of the essential and well-studied secretion systems which transport various virulence factors, including EspB. EspB, a ~36 kDa secreted substrate, has been implicated to play vital role in protecting the bacteria from hostile environment within the host cell phagosome. It is also involved in bacterial pathogenesis and has been shown to bind phospholipids. Recently, two cryo-EM structures of EspB full-length and the secreted isoforms were resolved. Despite the availability of multiple high-resolution structures of EspB, the physiological relevance and mechanism of virulence of this secreted substrate remains poorly characterized. In this current work, we implemented cryo-EM-based structural studies, including various functional assays, TEM imaging, and biophysical approach to demonstrate the interaction of EspB with lipids and bio-membrane. Our findings also indicated that EspB may play a crucial role in binding to and rupturing host mitochondrial membrane. Through cryo-EM studies we were able to show the possible membrane-binding region of EspB. Our study sheds light on host-pathogen interactions and bacterial pathogenesis mediated by EspB.
Structure and Epitope of a Neutralizing Monoclonal Antibody that Targets the Stem Helix of β Coronaviruses
Authors: Deshpande Ashlesha , Schormann Nobert , Piepenbrink Mike S. , Sobrido Luis Martinez , Kobie James J. , Walter Mark R.
Published: September 2022

Monoclonal antibodies (MAbs) that retain neutralizing activity against distinct coronavirus (CoV) lineages and variants of concern (VoC) must be developed to protect against future pandemics. These broadly neutralizing MAbs (BNMAbs) may be used as therapeutics and/or to assist in the rational design of vaccines that induce BNMAbs. 1249A8 is a BNMAb that targets the stem helix (SH) region of CoV spike (S) protein and neutralizes Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) original strain, delta, and omicron VoC, Severe Acute Respiratory Syndrome CoV (SARS-CoV) and Middle East Respiratory Syndrome CoV (MERS-CoV). To understand its mechanism of action, the crystal structure of 1249A8 bound to a MERS-CoV SH peptide was determined at 2.1Å resolution. BNMAb 1249A8 mimics the SARS-CoV-2 S loop residues 743-749, which interact with the C-terminal end of the SH helix in the S postfusion conformation. The crystal structure shows that BNMAb 1249A8 disrupts SH secondary structure and packing rearrangements required for CoV S to adopt its prefusion conformation that mediates membrane fusion and ultimately infection. The mechanisms regulating BNMAb 1249A8 CoV S specificity are also defined. This study provides novel insights into the neutralization mechanisms of SH-targeting CoV BNMAbs that may inform vaccine development and the design of optimal BNMAb therapeutics.
Microsecond melting and revitrification of cryo samples with a correlative light-electron microscopy approach
Authors: Bongiovanni Gabriele , Harder Oliver F. , Drabbels Marcel , Lorenz Ulrich J.
Published: September 2022

We have recently introduced a novel approach to time-resolved cryo-electron microscopy (cryo-EM) that affords microsecond time resolution. It involves melting a cryo sample with a laser beam to allow dynamics of the embedded particles to occur. Once the laser beam is switched off, the sample revitrifies within just a few microseconds, trapping the particles in their transient configurations, which can subsequently be imaged to obtain a snap shot of the dynamics at this point in time. While we have previously performed such experiments with a modified transmission electron microscope, we here demonstrate a simpler implementation that uses an optical microscope. We believe that this will make our technique more easily accessible and hope that it will encourage other groups to apply microsecond time-resolved cryo-EM to study the fast dynamics of a variety of proteins.
Systematic mutagenesis reveals critical effector functions in the assembly and dueling of the H1-T6SS inPseudomonas aeruginosa
Authors: Wu Li-Li , Pei Tong-Tong , Yan Shuangquan , Tang Ming-Xuan , Wu Li-Wen , Li Hao , Liang Xiaoye , Sun Shuyang , Dong Tao
Published: September 2022

Pseudomonas aeruginosais an important human pathogen that can cause severe wound and lung infections. It employs the type VI secretion system (H1-T6SS) as a molecular weapon to carry out a unique dueling response to deliver toxic effectors to neighboring sister cells or other microbes after sensing an external attack. However, the underlying mechanism for such dueling is not fully understood. Here, we examined the role of all H1-T6SS effectors and VgrG proteins in assembly and signal sensing by ectopic expression, combinatorial deletion and point mutations, and imaging analyses. Expression of effectors targeting the cell wall and membrane resulted in increased H1-T6SS assembly. Deletion of individual effector andvgrGgenes had minor- to-moderate effects on H1-T6SS assembly and dueling activities. The dueling response was detectable in theP. aeruginosamutant lacking all H1-T6SS effector activities. In addition, double deletions ofvgrG1awith eithervgrG1borvgrG1cand double deletions of effector genestse5andtse6severely reduced T6SS assembly and dueling activities, suggesting their critical role in T6SS assembly. Collectively, these data highlight the diverse roles of effectors in not only dictating antibacterial functions but also their differential contributions to the assembly of the complex H1-T6SS apparatus.
Perilipin 3 promotes the formation of membrane domains enriched in diacylglycerol and lipid droplet biogenesis proteins
Authors: Khaddaj Rasha , Schneiter Roger
Published: September 2022

Lipid droplets (LDs) serve as intracellular stores of energy-rich neutral lipids. LDs form at discrete sites in the endoplasmic reticulum (ER) and they remain closely associated with the ER during lipogenic growth and lipolytic consumption. Their hydrophobic neutral lipid core is covered by a monolayer of phospholipids, which harbors a specific set of proteins. This LD surface is coated and stabilized by perilipins, a family of soluble proteins that specifically target LDs from the cytosol. We have previously used chimeric fusion proteins between perilipins and integral ER membrane proteins to test whether proteins that are anchored to the ER bilayer could be dragged onto the LD monolayer. Expression of these chimeric proteins induces repositioning of the ER membrane around LDs. Here, we test the properties of membrane-anchored perilipins in cells that lack LDs. Unexpectedly, membrane-anchored perilipins induce the formation of crescent-shaped ER membrane domains that have LD-like properties. They are stained by lipophilic dyes and harbor LD marker proteins. In addition, these ER domains are enriched in diacylglycerol (DAG) and in ER proteins that are important for early steps of LD biogenesis, including seipin and Pex30. These ER membrane domains dissolve upon induction of neutral lipid synthesis and the formation of nascent LDs. Formation of these membrane domains in vivo requires DAG, and we show that perilipin 3 (PLIN3) binds to liposomes containing DAG in vitro. Taken together, these observations indicate that perilipin not only serve to stabilize the surface of mature LDs but that they are likely to exert a more active role in early steps of LD biogenesis at ER subdomains enriched in DAG, seipin, and neutral lipid biosynthetic enzymes.
A Second Role for the Second Messenger Cyclic-di-GMP inE. coli: Arresting Cell Growth by Altering Metabolic Flow
Authors: Hwang YuneSahng , Harshey Rasika M.
Published: September 2022

C-di-GMP primarily controls motile to sessile transitions in bacteria. Diguanylate cyclases (DGCs) catalyze the synthesis of c-di-GMP from two GTP molecules. Typically, bacteria encode multiple DGCs that are activated by specific environmental signals. Their catalytic activity is modulated by c-di-GMP binding to autoinhibitory sites (I-sites). YfiN is a conserved inner membrane DGC that lacks these sites. Instead, YfiN activity is directly repressed by periplasmic YfiR, which is inactivated by redox stress. InE. coli, an additional envelope stress causes YfiN to relocate to the mid-cell to inhibit cell division by interacting with the division machinery. Here, we report a third activity for YfiN inE. coli, where cell growth is inhibited without YfiN relocating to the division site. This action of YfiN is only observed when the bacteria are cultured on gluconeogenic carbon sources, and is dependent on absence of the autoinhibitory sites. Restoration of I-site function relieves the growth-arrest phenotype, and disabling this function in a heterologous DGC causes acquisition of this phenotype. Arrested cells are tolerant to a wide range of antibiotics. We show that the likely cause of growth arrest is depletion of cellular GTP from run-away synthesis of c-di-GMP, explaining the dependence of growth arrest on gluconeogenic carbon sources that exhaust more GTP during production of glucose. This is the first report of c-di-GMP-mediated growth arrest by altering metabolic flow.

Significance

The c-di-GMP signaling network in bacteria not only controls a variety of cellular processes such as motility, biofilms, cell development and virulence, but does so by a dizzying array of mechanisms. The DGC YfiN singularly represents the versatility of this network in that it not only inhibits motility and promotes biofilms, but also arrests growth inE. coliby relocating to the mid-cell and blocking cell division. The work described here reveals that YfiN arrests growth by yet another mechanism inE. coli– changing metabolic flow. This function of YfiN, or of DGCs without autoinhibitory I-sites, may contribute to antibiotic tolerant persisters in relevant niches such as the gut where gluconeogenic sugars are found.