bluuebunny/crossref_metadata_2025 huggingface dataset:
Non-Invasive Perfusion MR Imaging of the Human Brain via Breath-Holding
Authors: Schulman J.B. , Kashyap S. , Kim S.G. , Uludağ K.
Published: August 2023

Dynamic susceptibility contrast (DSC) MRI plays a pivotal role in the accurate diagnosis and prognosis of several neurovascular diseases, but is limited by its reliance on gadolinium, an intravascularly injected chelated metal. Here, we determined the feasibility of measuring perfusion using a DSC analysis of breath-hold-induced gradient-echo-MRI signal changes. We acquired data at both 3T and 7T from ten healthy participants who engaged in eight consecutive breath-holds. By pairing a novel arterial input function strategy with a standard DSC MRI analysis, we measured the cerebral blood volume, flow, and transit delay, and found values to agree with those documented in the literature using gadolinium. We also observed voxel-wise agreement between breath-hold and arterial spin labeling measures of cerebral blood flow. Breath-holding resulted in significantly higher contrast-to-noise (6.2 at 3T vs 8.5 at 7T) and gray matter-to-white matter contrast at higher field strength. Finally, using a simulation framework to assess the effect of dynamic vasodilation on perfusion estimation, we found global perfusion underestimation of 20-40%. For the first time, we have assessed the feasibility of and limitations associated with using breath-holds for perfusion estimation with DSC. We hope that the methods and results presented in this study will help pave the way toward contrast-free perfusion imaging, in both basic and clinical research.
Heroes and villains: opposing narrative roles engage neural synchronization in the lateral inferior frontal gyrus
Authors: Ryu Hayoung , Kim M. Justin
Published: August 2023

Neuroscientific studies have highlighted the role of the default mode network (DMN) in processing narrative information. Here, we examined whether the neural synchronization of the DMN tracked the appearances of characters with different narrative roles (i.e., protagonists versus antagonists) when viewing highly engaging, socially rich audiovisual narratives. Using inter-subject correlation analysis on two independent, publicly available movie-watching functional magnetic resonance imaging datasets (SherlockandThe Grand Budapest Hotel), we computed whole-brain neural synchronization during the appearance of the protagonists and antagonists. Results showed that the inferior frontal gyrus (IFG) and orbitofrontal cortex (OFC), which are components of the DMN, had higher ISC values during the appearance of the protagonists compared to the antagonists. Importantly, these findings were commonly observed in both datasets. We discuss the present results in the context of information integration and emotional empathy, which are relevant functions known to be supported by the DMN. Our study presents generalizable evidence that regions within the DMN – particularly the IFG and OFC – show distinctive synchronization patterns due to differences in narrative roles.
A hybrid machine learning and regression method for cell type deconvolution of spatial barcoding-based transcriptomic data
Authors: Liu Yunqing , Li Ningshan , Qi Ji , Xu Gang , Zhao Jiayi , Wang Nating , Huang Xiayuan , Jiang Wenhao , Justet Aurélien , Adams Taylor S. , Homer Robert , Amei Amei , Rosas Ivan O. , Kaminski Naftali , Wang Zuoheng , Yan Xiting
Published: August 2023

Spatial barcoding-based transcriptomic (ST) data require cell type deconvolution for cellular-level downstream analysis. Here we present SDePER, a hybrid machine learning and regression method, to deconvolve ST data using reference single-cell RNA sequencing (scRNA-seq) data. SDePER uses a machine learning approach to remove the systematic difference between ST and scRNA-seq data (platform effects) explicitly and efficiently to ensure the linear relationship between ST data and cell type-specific expression profile. It also considers sparsity of cell types per capture spot and across-spots spatial correlation in cell type compositions. Based on the estimated cell type proportions, SDePER imputes cell type compositions and gene expression at unmeasured locations in a tissue map with enhanced resolution. Applications to coarse-grained simulated data and four real datasets showed that SDePER achieved more accurate and robust results than existing methods, suggesting the importance of considering platform effects, sparsity and spatial correlation in cell type deconvolution.
Quantitative image analysis pipeline for detecting circulating hybrid cells in immunofluorescence images with human-level accuracy
Authors: Heussner Robert T. , Whalen Riley M. , Anderson Ashley , Theison Heather , Baik Joseph , Gibbs Summer , Wong Melissa H. , Chang Young Hwan
Published: August 2023

Circulating hybrid cells (CHCs) are a newly discovered, tumor-derived cell population identified in the peripheral blood of cancer patients and are thought to contribute to tumor metastasis. However, identifying CHCs by immunofluorescence (IF) imaging of patient peripheral blood mononuclear cells (PBMCs) is a time-consuming and subjective process that currently relies on manual annotation by laboratory technicians. Additionally, while IF is relatively easy to apply to tissue sections, its application on PBMC smears presents challenges due to the presence of biological and technical artifacts. To address these challenges, we present a robust image analysis pipeline to automate the detection and analyses of CHCs in IF images. The pipeline incorporates quality control to optimize specimen preparation protocols and remove unwanted artifacts, leverages a β-variational autoencoder (VAE) to learn meaningful latent representations of single-cell images and employs a support vector machine (SVM) classifier to achieve human-level CHC detection. We created a rigorously labeled IF CHC dataset including 9 patients and 2 disease sites with the assistance of 10 annotators to evaluate the pipeline. We examined annotator variation and bias in CHC detection and then provided guidelines to optimize the accuracy of CHC annotation. We found that all annotators agreed on CHC identification for only 65% of the cells in the dataset and had a tendency to underestimate CHC counts for regions of interest (ROI) containing relatively large amounts of cells (>50,000) when using conventional enumeration methods. On the other hand, our proposed approach is unbiased to ROI size. The SVM classifier trained on the β-VAE encodings achieved an F1 score of 0.80, matching the average performance of annotators. Our pipeline enables researchers to explore the role of CHCs in cancer progression and assess their potential as a clinical biomarker for metastasis. Further, we demonstrate that the pipeline can identify discrete cellular phenotypes among PBMCs, highlighting its utility beyond CHCs.
Mutational analysis of the nitrogenase carbon monoxide protective protein CowN reveals that a conserved C-terminal glutamic acid residue is necessary for its activity
Authors: Willard Dustin L. , Arellano Joshuah J. , Underdahl Mitch , Lee Terrence M. , Ramaswamy Avinash S. , Fumes Gabriella , Kliman Agatha , Wong Emily Y. , Owens Cedric P.
Published: August 2023

Nitrogenase is the only enzyme that catalyzes the reduction of nitrogen gas into ammonia. Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Many nitrogen fixing bacteria protect nitrogenase from CO inhibition using the protective protein CowN. This work demonstrates that a conserved glutamic acid residue near CowN’s C-terminus is necessary for its function. Mutation of the glutamic acid residue abolishes both CowN’s protection against CO inhibition and CowN’s ability to bind to nitrogenase. In contrast, a conserved C-terminal cysteine residue is not important for CO protection. Overall, this work uncovers structural features in CowN that are required for its function and provides new insights into its nitrogenase binding and CO protection mechanism.
Tribbles1 and Cop1 cooperate to protect the host duringin vivomycobacterial infection
Authors: Hammond Ffion R , Lewis Amy , Pollara Gabriele , Tomlinson Gillian S , Noursadeghi Mahdad , Kiss-Toth Endre , Elks Philip M
Published: August 2023

Tuberculosis is a major global health problem and is one of the top 10 causes of death worldwide. There is a pressing need for new treatments that circumvent emerging antibiotic resistance.Mycobacterium tuberculosisparasitises macrophages, reprogramming them to establish a niche in which to proliferate, therefore macrophage manipulation is a potential host-directed therapy if druggable molecular targets could be identified. The pseudokinase Tribbles1 (Trib1) regulates multiple innate immune processes and inflammatory profiles making it a potential drug target in infections. Trib1 controls macrophage function, cytokine production and macrophage polarisation. Despite wide-ranging effects on leukocyte biology, data exploring the roles of Tribbles in infectionin vivoare limited. Here, we identify that human Tribbles 1 is expressed in monocytes and is upregulated at the transcript level after stimulation with mycobacterial antigen. To investigate the mechanistic roles of Tribbles in the host response to mycobacteriain vivo, we used a zebrafishMycobacterium marinum(Mm) infection tuberculosis model. Zebrafish Tribbles family members were characterised and shown to have substantial mRNA and protein sequence homology to their human orthologues.trib1overexpression was host-protective against Mm infection, reducing burden by approximately 50%. Conversely,trib1knockdown exhibited increased infection. Mechanistically,trib1overexpression significantly increased the levels of pro-inflammatory factorsil-1β and nitric oxide. The host-protective effect oftrib1was found to be dependent on the E3 ubiquitin kinase Cop1. These findings highlight the importance of Trib1 and Cop1 as immune regulators during infectionin vivoand suggest that enhancing macrophage TRIB1 levels may provide a tractable therapeutic intervention to improve bacterial infection outcomes in tuberculosis.
Identification of clade-defining single nucleotide polymorphisms for improved rabies virus surveillance
Authors: Kumar Ankeet , Tushir Sheetal , Devasurmutt Yashas , Nath Sujith S , Tatu Utpal
Published: August 2023

Background

Rabies is an ancient disease that remains endemic in many countries. It causes many human deaths annually, predominantly in resource-poor countries. Over evolutionary timelines, several rabies virus (RABV) genotypes have stabilised, forming distinct clades. Extensive studies have been conducted on the origin, occurrence and spread of RABV clades. Single nucleotide polymorphisms (SNPs) distribution across the RABV genome and its clades remains largely unknown, highlighting the need for comprehensive whole-genome analyses.

Methods

We accessed whole genome sequences for RABV from public databases and identified SNPs across the whole genome sequences. Then, we annotated these SNPs using an R script, and these SNPs were categorised into different categories; universal, clade-specific, and clade-defining, based on the frequency of occurrence.

Results

In this study, we present the SNPs occurring in the RABV based on whole genome sequences belonging to 8 clades isolated from 7 different host species likely to harbour dog-related rabies. We classified mutations into several classes based on their location within the genome and assessed the effect of SNP mutations on the viral glycoprotein.

Conclusions

The clade-defining mutations have implications for targeted surveillance and classification of clades. Additionally, we investigated the effects of these mutations on the Glycoprotein of the virus. Our findings contribute to expanding knowledge about RABV clade diversity and evolution, which has significant implications for effectively tracking and combatting RABV transmission.
Phylogenetically diverse wild plant species use common biochemical strategies to thrive in the Atacama Desert
Authors: Dussarrat Thomas , Nilo-Poyanco Ricardo , Moyano Tomás C. , Prigent Sylvain , Jeffers Tim L. , Díaz Francisca P. , Decros Guillaume , Audi Lauren , Sondervan Veronica M , Shen Bingran , Araus Viviana , Rolin Dominique , Shasha Dennis , Coruzzi Gloria M. , Gibon Yves , Latorre Claudio , Pétriacq Pierre , Gutiérrez Rodrigo A.
Published: August 2023

The best ideotypes are under mounting pressure due to increased aridity in many parts of the world. Understanding the conserved molecular mechanisms that evolve in wild plant species adapted to harsh environments is crucial in developing new strategies for sustainable agriculture. Yet our knowledge of such mechanisms in wild species is scant, particularly in extreme environments. We performed metabolic pathway reconstruction using transcriptome information from 32 Atacama plant species and phylogenetically related plant species that do not live in Atacama (Sister species). We analyzed pathway and reaction enrichment to understand the biochemical commonalities and differences of wild Atacama plant species. To gain insights into the mechanisms that ensure plant survival, we compared expressed gene isoform numbers and gene expression patterns between the annotated biochemical reactions from 32 Atacama and Sister species. We found significant biochemical convergences in primary and secondary metabolism characterized by reactions enriched in at least 50% of the Atacama species across major plant phylogenetic lineages. Analysis of the annotation indicated potential advantages against drought, salinity, high solar irradiance, and nitrogen starvation. These findings suggest that the adaptation in the Atacama Desert may result in part from shared genetic legacies governing the expression of key metabolic pathways to face harsh environmental conditions. Enriched reactions corresponded to ubiquitous compounds common to extreme and agronomic species and were congruent with our previous metabolomic analyses in these Atacama species. Hence, genes underlying these adaptive traits offer promising candidates for improving abiotic stress resilience in crop species.
4D intravital imaging studies identify platelets as the predominant cellular procoagulant surface in a mouse model of hemostasis
Authors: Ballard-Kordeliski Abigail , Lee Robert H. , O’Shaughnessy Ellen C. , Kim Paul Y. , Jones Summer , Mackman Nigel , Flick Matthew J. , Paul David S. , Adalsteinsson David , Bergmeier Wolfgang
Published: August 2023

Interplay between platelets, coagulation/fibrinolytic factors, and endothelial cells (ECs) is necessary for effective hemostatic plug formation. This study describes a novel four-dimensional (4D) imaging platform to visualize and quantify hemostatic plug components with high spatiotemporal resolution. Fibrin accumulation following laser-induced endothelial ablation was observed at the EC-platelet plug interface, controlled by the antagonistic balance between fibrin generation and breakdown. Phosphatidylserine (PS) was first detected in close physical proximity to the fibrin ring, followed by exposure across the endothelium. Impaired PS exposure incyclophilinD-/-mice resulted in a significant reduction in fibrin accumulation. Adoptive transfer and inhibitor studies demonstrated a key role for platelets, but not ECs, in fibrin generation during hemostatic plug formation. Inhibition of fibrinolysis with tranexamic acid (TXA) led to increased fibrin accumulation in WT mice, but not incyclophilinD-/-mice or WT mice treated with antiplatelet drugs. These studies implicate platelets as the functionally dominant procoagulant surface during hemostatic plug formation. In addition, they suggest that impaired fibrin formation due to reduced platelet procoagulant activity is not reversed by TXA treatment.
The cGAS-STING pathway regulates microglial chemotaxis in genome instability
Authors: Talbot Emily J. , Joshi Lisha , Thornton Peter , Dezfouli Mahya , Tsafou Kalliopi , Perkinton Michael , Khoronenkova Svetlana V.
Published: August 2023

Defective DNA damage signalling and repair is a hallmark of age-related and genetic neurodegenerative disease. One mechanism implicated in disease progression is DNA damage-driven neuroinflammation, which is largely mediated by tissue-resident immune cells, microglia. Here, we utilise human microglia-like cell models of persistent DNA damage and ATM kinase deficiency to investigate how genome instability shapes microglial function. We demonstrate that upon DNA damage the cytosolic DNA sensing cGAS-STING axis drives chronic inflammation and a robust chemokine response, exemplified by production of CCL5 and CXCL10. Transcriptomic analyses revealed that cell migratory pathways were highly enriched upon IFN-β treatment of human iPSC-derived microglia, indicating that the chemokine response to DNA damage mirrors type I interferon signalling. Furthermore, we find that STING deletion leads to a defect in microglial chemotaxis under basal conditions and upon ATM kinase loss. Overall, this work provides mechanistic insights into cGAS-STING-dependent neuroinflammatory mechanisms and consequences of genome instability in the central nervous system.
A single base pair substitution on Chromosome 25 in zebrafish distinguishes between development and acute regulation of behavioral thresholds
Authors: Ortiz Elelbin A. , Campbell Philip D. , Nelson Jessica C. , Granato Michael
Published: August 2023

Behavioral thresholds define the lowest stimulus intensities sufficient to elicit a behavioral response. Establishment of baseline behavioral thresholds during development is critical for proper responses throughout the animal’s life. Despite the relevance of such innate thresholds, the molecular mechanisms critical to establishing behavioral thresholds during development are not well understood. The acoustic startle response is a conserved behavior whose threshold is established during development yet is subsequently acutely regulated. We have previously identified a zebrafish mutant line (escapist) that displays a decreased baseline or innate acoustic startle threshold. Here, we identify a single base pair substitution on Chromosome 25 located within the coding sequence of thesynaptotagmin 7a(syt7a) gene that is tightly linked to theescapistacoustic hypersensitivity phenotype. By generating animals in which we deleted thesyt7aopen reading frame, and subsequent complementation testing with theescapistline, we demonstrate that loss ofsyt7afunction is not the cause of theescapistbehavioral phenotype. Nonetheless,escapistmutants provide a powerful tool to decipher the overlap between acute and developmental regulation of behavioral thresholds. Extensive behavioral analyses reveal that inescapistmutants the establishment of the innate acoustic startle threshold is impaired, while regulation of its acute threshold remains intact. Moreover, our behavioral analyses reveal a deficit in baseline responses to visual stimuli, but not in the acute regulation of responses to visual stimuli. Together, this work eliminates loss ofsyt7aas causative for theescapistphenotype and suggests that mechanisms that regulate the establishment of behavioral thresholds inescapistlarvae can operate largely independently from those regulating acute threshold regulation.
Imaging analysis of six human histone H1 variants reveals universal enrichment of H1.2, H1.3, and H1.5 at the nuclear periphery and nucleolar H1X presence
Authors: Salinas-Pena Mónica , Rebollo Elena , Jordan Albert
Published: August 2023

Histone H1 participates in chromatin condensation and regulates nuclear processes. Human somatic cells may contain up to seven histone H1 variants, although their functional heterogeneity is not fully understood. Here, we have profiled the differential nuclear distribution of the somatic H1 repertoire in human cells through imaging techniques including super-resolution microscopy. H1 variants exhibit characteristic distribution patterns in both interphase and mitosis. H1.2, H1.3, and H1.5 are universally enriched at the nuclear periphery in all cell lines analyzed and co-localize with compacted DNA. H1.0 shows a less pronounced peripheral localization, with apparent variability among different cell lines. On the other hand, H1.4 and H1X are distributed throughout the nucleus, being H1X universally enriched in high-GC regions and abundant in the nucleoli. Interestingly, H1.4 and H1.0 show a more peripheral distribution in cell lines lacking H1.3 and H1.5. The differential distribution patterns of H1 suggest specific functionalities in organizing lamina-associated domains or nucleolar activity, which is further supported by a distinct response of H1X or phosphorylated H1.4 to the inhibition of rDNA transcription. Moreover, H1 variants depletion affects chromatin structure in a variant-specific manner. Concretely, H1.2 knock-down, either alone or combined, triggers a global chromatin decompaction. Overall, imaging has allowed us to distinguish H1 variants distribution beyond the segregation in two groups denoted by previous ChIP-seq determinations. Our results support H1 variants heterogeneity and suggest that variant-specific functionality can be shared between different cell types.
Local and dynamic regulation of neuronal glycolysisin vivo
Authors: Wolfe Aaron D. , Koberstein John N , Smith Chadwick B , Stewart Melissa L , Hammarlund Marc , Hyman Anthony , Stork Philip JS , Goodman Richard , Colón-Ramos Daniel A.
Published: August 2023

Energy metabolism supports neuronal function. While it is well established that changes in energy metabolism underpin brain plasticity and function, less is known about how individual neurons modulate their metabolic states to meet varying energy demands. This is because most approaches used to examine metabolism in living organisms lack the resolution to visualize energy metabolism within individual circuits, cells, or subcellular regions. Here we adapted a biosensor for glycolysis, HYlight, for use inC. elegansto image dynamic changes in glycolysis within individual neurons andin vivo. We determined that neurons perform glycolysis cell-autonomously, and modulate glycolytic states upon energy stress. By examining glycolysis in specific neurons, we documented a neuronal energy landscape comprising three general observations: 1) glycolytic states in neurons are diverse across individual cell types; 2) for a given condition, glycolytic states within individual neurons are reproducible across animals; and 3) for varying conditions of energy stress, glycolytic states are plastic and adapt to energy demands. Through genetic analyses, we uncovered roles for regulatory enzymes and mitochondrial localization in the cellular and subcellular dynamic regulation of glycolysis. Our study demonstrates the use of a single-cell glycolytic biosensor to examine how energy metabolism is distributed across cells and coupled to dynamic states of neuronal function, and uncovers new relationships between neuronal identities and metabolic landscapesin vivo.

Significance statement

While it is generally accepted that energy metabolism underpins neuronal function, how it is distributed and dynamically regulated in different tissues of the brain to meet varying energy demands is not well understood. Here we utilized a fluorescent biosensor, HYlight, to observe glycolytic metabolism at cellular and subcellular scalesin vivo. By leveraging both the stereotyped identities of individual neurons inC. elegans,and genetic tools for manipulating glycolytic metabolism, we determined that neurons perform and dynamically regulate glycolysis to match changing cellular demands for energy. Our findings support a model whereby glycolytic states should be considered distinct and related to individual neuron identitiesin vivo, and introduce new questions about the interconnected nature of metabolism and neuronal function.
Single-molecule visualization of twin-supercoiled domains generated during transcription
Authors: Janissen Richard , Barth Roman , Polinder Minco , van der Torre Jaco , Dekker Cees
Published: August 2023

Transcription-coupled supercoiling of DNA is a key factor in chromosome compaction and the regulation of genetic processes in all domains of life. It has become common knowledge that, during transcription, the DNA-dependent RNA polymerase (RNAP) induces positive supercoiling ahead of it (downstream) and negative supercoils in its wake (upstream), as rotation of RNAP around the DNA axis upon tracking its helical groove gets constrained due to drag on its RNA transcript. Here, we experimentally validate this so-called twin-supercoiled-domain model within vitroreal-time visualization at the single-molecule scale. Upon binding to the promoter site on a supercoiled DNA molecule, RNAP merges all DNA supercoils into one large pinned plectoneme with RNAP residing at its apex. Transcription by RNAP in real time demonstrates that up- and downstream supercoils are generated simultaneously and in equal portions, in agreement with the twin-supercoiled-domain model. Experiments carried out in the presence of RNases A and H, revealed that an additional viscous drag of the RNA transcript is not necessary for the RNAP to induce supercoils. The latter results contrast the current consensus and simulations on the origin of the twin-supercoiled domains, pointing at an additional mechanistic cause underlying supercoil generation by RNAP in transcription.
Disease Resistance correlates with Core Microbiome Diversity in Cotton
Authors: Aqueel Rhea , Badar Ayesha , Roy Nazish , Ijaz Umer Zeeshan , Malik Kauser Abdulla
Published: August 2023

Understanding the resident microbial communities and their above and below ground interactions with plants will provide necessary information for crop disease protection and stress management. In this study, we show how diversity of core microbiome varies with disease susceptibility of a crop. To test this hypothesis, we have focused on identifying the core microbial species of Cotton Leaf Curl Disease (CLCuD) susceptibleGossypium hirsutumand CLCuD resistantGossypium arboreumunder viral infestation. Derivation of core membership is challenging as it depends on an occupancy threshold of microbial species in a sampling pool, whilst accounting for different plant compartments. We have used an abundance-occupancy distribution approach where we dynamically assess the threshold for core membership, whilst marginalizing for occupancy in four compartments of the cotton plant, namely, leaf epiphyte, leaf endophyte, rhizosphere, and root endophyte. Additionally, we also fit a neutral model to the returned core species to split them into three groups, those that are neutral, those that are selected by the plant environment, and finally those that are dispersal limited. We have found strong inverse relationship between diversity of core microbiome and disease susceptibility. A deeper understanding of this association will aid in the development of biocontrol agents for improving plant immunity against biotrophic pathogens.
Heat shock induces alternative polyadenylation through dynamic DNA methylation-regulated chromatin looping
Authors: Fink Emily E. , Nanavaty Vishal , Lee Byron H. , Ting Angela H.
Published: August 2023

Alternative cleavage and polyadenylation (APA) is a gene regulatory mechanism used by cells under stress to upregulate proteostasis-promoting transcripts, but how cells achieve this remains poorly understood. Previously, we elucidated a DNA methylation-regulated APA mechanism, in which gene body DNA methylation enhances distal poly(A) isoform expression by blocking CTCF binding and chromatin loop formation at APA control regions. We hypothesized that DNA methylation-regulated APA is one mechanism cells employ to induce proteostasis-promoting poly(A) isoforms. At theDNAJB6co-chaperone gene locus, acute heat shock resulted in binding of stress response transcription factors HSF1, ATF6, and YY1 at the APA control region and an increase in the expression of the proximal poly(A) isoform known to prevent protein aggregation. Furthermore, TET1 was recruited to rapidly demethylate DNA, facilitating CTCF binding and chromatin loop formation, thereby reinforcing preferential proximal poly(A) isoform expression. As cells recovered, the transcription factors vacated the APA control region, and DNMT1 was recruited to remethylate the region. This process resolved chromatin looping and reset the poly(A) isoform expression pattern. Our findings unveil an epigenetic mechanism enabling cells to dynamically modulate poly(A) isoforms in response to stress while shedding light on the interplay between DNA methylation, transcription factors, and chromatin looping.
CryoEM architecture of a native stretch-sensitive membrane microdomain
Authors: Kefauver Jennifer M. , Hakala Markku , Zou Luoming , Alba Josephine , Espadas Javier , Tettamanti Maria G. , Estrozi Leandro F. , Vanni Stefano , Roux Aurélien , Desfosses Ambroise , Loewith Robbie
Published: August 2023

Biological membranes are partitioned into functional zones containing specific lipids and proteins, termed membrane microdomains. Their composition and organization remain controversial owing to a paucity of techniques that can visualize lipidsin situwithout disrupting their native behavior1,2. The yeast eisosome, a membrane compartment scaffolded by the BAR-domain proteins Pil1 and Lsp1, senses and responds to mechanical stress by flattening and releasing sequestered factors3–7. Here, we isolated native eisosomes as helical filaments of Pil1/Lsp1 lattice bound to plasma membrane lipids and solved their structures by helical reconstruction. We observe remarkable organization within the lipid bilayer density from which we could assign headgroups of PI(4,5)P2and phosphatidylserine bound to Pil1/Lsp1 and a pattern of membrane voids, signatures of sterols, beneath an amphipathic helix. We verified these assignments usingin vitroreconstitutions and molecular dynamics simulations. 3D variability analysis of the native eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects functionally important lipid sequestration, supporting a mechanism in which membrane stretching liberates lipids otherwise anchored by the Pil1/Lsp1 coat. Our results provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain and, more globally, resolve long-standing controversies about the architecture and nature of lipid microdomains.
An ultraviolet-driven rescue pathway for oxidative stress to eye lens protein human gamma-D crystallin
Authors: Hill Jake A , Nyathi Yvonne , Horrell Sam , von Stetten David , Axford Danny , Owen Robin L , Beddard Godfrey , Pearson Arwen R , Ginn Helen Mary , Yorke Briony A
Published: August 2023

1

Human gamma-D crystallin (HGD) is the major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin shows no post-translational modifications. After aging for nine months in the absence of light, a covalently bound reducing agent modifying surface cysteines is observed for the first time by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.
Cdc42 couples septin recruitment to the axial landmark assembly via Axl2 in budding yeast
Authors: Kang Pil Jung , Mullner Rachel , Lian Kendra , Park Hay-Oak
Published: August 2023

Cell polarization generally occurs along a single axis that is directed by a spatial cue. Cells of the budding yeastSaccharomyces cerevisiaeundergo polarized growth and oriented cell division in a spatial pattern by selecting a specific bud site. Haploidaor α cells bud in the axial pattern in response to a transient landmark that includes Bud3, Bud4, Axl1, and Axl2. Septins, a family of filament-forming GTP-binding proteins, are also involved in axial budding and recruited to an incipient bud site, but the mechanism of recruitment remains unclear. Here, we show that Axl2 interacts with Bud3 and the Cdc42 GTPase in its GTP-bound state. Axl2 also interacts with Cdc10, a septin subunit, promoting efficient recruitment of septins near the cell division site. Furthermore, acdc42mutant defective in the axial budding pattern at a semi-permissive temperature had a reduced interaction with Axl2 and compromised septin recruitment in the G1 phase. We thus propose that active Cdc42 brings Axl2 to the Bud3-Bud4 complex and that Axl2 then interacts with Cdc10, linking septin recruitment to the axial landmark.
Automappa: An interactive interface for metagenome-derived genome bins
Authors: Rees Evan R. , Waterworth Samantha C. , Chanana Shaurya C. , Kwan Jason C.
Published: August 2023

Background

Studies attempting to observe microbes commonly considered uncultivable under standard laboratory conditions are turning to so-called “deep” environmental sequencing approaches whereby they may access these unculturable organisms’ genomes viain silicoapproaches. A typical workflow involves metagenome assembly, annotation, and binning for reconstruction of each respective organism’s genome (or metagenome-assembled genome, MAG). Many automated genome binning approaches have been developed and have displayed a wide range of variation in performance. Therefore, refinement methods have been developed in order to aid manual curation following the automated genome binning process. Current manual curation tools were developed with a focus towards teaching metagenomics concepts and may fail when handling complex datasets containing many microbes. Automappa was developed with a focus on overlaying a variety of annotations such as taxonomy, coverage and marker-gene prevalence while maintaining an implementation that may scale to the complexity of environmental samples.

Results

We present Automappa, a companion tool and interactive interface for exploration and refinement of Autometa taxon and genome binning results from metagenomes. Selections provide real-time updates of MAG metrics to aid manual curation. Furthermore, researchers may detect unbinned MAGs as well as manually improve their draft-quality MAGs with contigs that closely match the MAG’s genome characteristics. Automappa’s utility has previously been demonstrated on host-associated, marine and terrestrial systems with a total of 242 curated MAGs across fourteen published metagenomes. Of these refined MAGs, the number of high-quality and medium-quality bins increased, consequently lowering the number of low-quality bins and decreasing the amount of data discarded from downstream analyses. The recovery of higher quality MAGs improved the confidence in results and strengthened the resultant conclusions of these respective studies. Automappa consists of three tabs, one for uploading a user’s metagenome data, another for exploration and refinement and the last for providing an overall summary of the refined MAG results.

Conclusions

Automappa is an open source software package that allows researchers to easily assess and refine undetected or draft-quality MAGs from their respective metagenomes. It is freely available under the GPLv3 license athttps://github.com/WiscEvan/Automappaand through Figshare (doi: 10.6084/m9.figshare.22593235).
Disturbance-Diversity Relationships of Microbial Communities Change Based on Growth Substrate
Authors: Hoang Don Q. , Wilson Lindsay R. , Scheftgen Andrew J. , Suen Garret , Currie Cameron R.
Published: August 2023

Disturbance events can impact ecological community dynamics. Understanding how communities respond to disturbances, and how those responses can vary, is a challenge in microbial ecology. In this study, we grew a previously enriched specialized microbial community on either cellulose or glucose as a sole carbon source, and subjected them to one of five different disturbance regimes of varying frequencies ranging from low to high. Using 16S rRNA gene amplicon sequencing, we show that community structure is largely driven by substrate, but disturbance frequency affects community composition and successional dynamics. When grown on cellulose, bacteria in the generaCellvibrio,Lacunisphaera, andAsticaccacaulisare the most abundant microbes. However,Lacunisphaerais only abundant in the lower disturbance frequency treatments, whileAsticaccaulisis more abundant in the highest disturbance frequency treatment. When grown on glucose, the most abundant microbes are twoPseudomonassequence variants, and aCohnellasequence variant that is only abundant in the highest disturbance frequency treatment. Communities grown on cellulose exhibited a greater range of diversity (0.67-1.99 Shannon diversity and 1.38-5.25 Inverse Simpson diversity) that peak at the intermediate disturbance frequency treatment, or 1 disturbance every 3 days. Communities grown on glucose, however, ranged from 0.49-1.43 Shannon diversity and 1.37-3.52 Inverse Simpson with peak diversity at the greatest disturbance frequency treatment. These results demonstrate that the dynamics of a microbial community can vary depending on substrate and the disturbance frequency, and may potentially explain the variety of diversity-disturbance relationships observed in microbial ecosystems.

Abstract Importance

A generalizable diversity-disturbance relationship (DDR) of microbial communities remains a contentious topic. Various microbial systems have different DDRs. Rather than finding support or refuting specific DDRs, we investigated the underlying factors that lead to different DDRs. In this study, we measured a cellulose-enriched microbial community’s response to a range of disturbance frequencies from high to low, across two different substrates: cellulose and glucose. We demonstrate that the community displays a unimodal DDR when grown on cellulose, and a monotonically increasing DDR when grown on glucose. Our findings suggest that the same community can display different DDRs. These results suggest that the range of DDRs we observe across different microbial systems may be due to the nutritional resources microbial communities can access and the interactions between bacteria and their environment.
Genome analyses reveal the secondary metabolites potentially influence the geographical distribution ofFusarium pseudograminearumpopulations
Authors: Li Wei , Cao Shulin , Sun Haiyan , Yang Xiaoyue , Xu Lei , Zhang Xin , Deng Yuanyu , Pavlov Igor N. , Litovka Yulia A. , Chen Huaigu
Published: August 2023

Fusarium crown rot (FCR), caused byFusarium pseudograminearum, significantly impacts wheat yield and quality in China’s Huanghuai region. The rapidF. pseudograminearumepidemic and FCR outbreak within a decade remain unexplained. In this study, two high-quality, chromosome-level genomes ofF. pseudograminearumstrains producing 3-acetyl-deoxynivalenol (3AcDON) and 15-acetyl-deoxynivalenol (15AcDON) toxins were assembled. Additionally, 38 related strains were resequenced. Genomic differences such as single nucleotide polymorphisms (SNPs), insertions/deletions (indels), and structural variations (SVs) amongF. pseudograminearumstrains were analyzed. The whole-genome SNP locus based population classification mirrored the toxin chenotype (3AcDON and 15AcDON)-based classification, indicating the presence of genes associated with the trichothecene toxin gene cluster. Further analysis of differential SNP, indel, and SV loci between the 3AcDON and 15AcDON populations revealed a predominant connection to secondary metabolite synthesis genes. Notably, the majority of the secondary metabolite biosynthesis gene cluster (SMGC) loci were located in SNP-dense genomic regions, suggesting high mutability and a possible contribution toF. pseudograminearumpopulation structure and environmental adaptability. This study provides insightful perspectives on the distribution and evolution ofF. pseudograminearum, and for forecasting the spread of wheat FCR, thereby aiding in the development of preventive measures and control strategies.
Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology
Authors: Filandr Frantisek , Sarpe Vladimir , Raval Shaunak , Alex Crowder D. , Khan Morgan F. , Douglas Pauline , Coales Stephen , Viner Rosa , Syed Aleem , Tainer John A. , Lees-Miller Susan P. , Schriemer David C.
Published: August 2023

We developed a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS2) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-validation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS1-based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy. The approach requires data-independent acquisition (DIA) methods that are available on most MS platforms, making the switch to HX-MS2straightforward. Importantly, we find that HX-DIA enables a proteomics-grade approach and wide-spread applications. Considerable time is saved through auto-validation and complex samples can now be characterized and at higher throughput. We illustrate these advantages in a drug binding analysis of the ultra-large protein kinase DNA-PKcs, isolated directly from mammalian cells.
The G protein alpha Chaperone and Guanine-Nucleotide Exchange Factor RIC-8 Regulates Cilia Morphogenesis inCaenorhabditis elegansSensory Neurons
Authors: Campagna Christina M. , McMahon Hayley , Nechipurenko Inna
Published: August 2023

Heterotrimeric G (αβγ) proteins are canonical transducers of G-protein-coupled receptor (GPCR) signaling and play critical roles in communication between cells and their environment. Many GPCRs and heterotrimeric G proteins localize to primary cilia and modulate cilia morphology via mechanisms that are not well understood. Here, we show that RIC-8, a cytosolic guanine nucleotide exchange factor (GEF) and chaperone for Gα protein subunits, shapes cilia membrane morphology in a subset ofCaenorhabditis eleganssensory neurons. Consistent with its role in ciliogenesis,C. elegansRIC-8 localizes to cilia in different sensory neuron types. Using domain mutagenesis, we demonstrate that while the GEF function alone is not sufficient, both the GEF and Gα-interacting chaperone motifs of RIC-8 are required for its role in cilia morphogenesis. We identify ODR-3 as the RIC-8 Gα client and demonstrate that RIC-8 functions in the same genetic pathway with another component of the non-canonical G protein signaling AGS-3 to shape cilia morphology. Notably, despite severe defects in AWC cilia morphology,ags-3null mutants exhibit normal chemotaxis toward benzaldehyde unlikeodr-3mutant animals. Collectively, our findings describe a novel function for the evolutionarily conserved protein RIC-8 and non-canonical RIC-8-AGS-3-ODR-3 signaling in cilia morphogenesis and uncouple Gα ODR-3 functions in ciliogenesis and olfaction.

Author Summary

Primary cilia are specialized cellular compartments that mediate communication between cells and their environment. In humans, deficits in cilia assembly and function lead to genetic diseases called ciliopathies. While the molecular machinery that sends out and receives major signaling cues is present inside cilia of different cell types, the mechanisms by which these signaling factors modulate cilia structure or cilia-dependent cellular functions are not well understood. In this study, we identify a new role for the RIC-8-AGS-3-ODR-3 signaling module in formation of specialized ciliary structures inC. elegansneurons. Our findings provide new insight into mechanisms of cilia assembly and highlight how the same signaling component (i.e. ODR-3) can be differentially regulated to mediate distinct cellular functions (cilia formation versus olfactory behavior).
Partnership with both fungi and bacteria can protectOdontotermes obesusfungus gardens against fungal invaders
Authors: Agarwal Renuka , Gupta Manisha , Sen Ruchira , E.S. Nimisha , Raychoudhury Rhitoban
Published: August 2023

Fungus-growing termites, likeOdontotermes obesus, cultivateTermitomycesas their sole food source on fungus combs which are continuously maintained with foraged plant materials. This necessary augmentation also increases the threat of introducing pathogenic fungi capable of displacingTermitomyces. The magnitude of this threat and how termites prevent pathogens remain largely unknown. This study identifies this pathogenic load by establishing the pan-mycobiota ofO. obesusfrom the fungus comb and termite castes. Furthermore, to maximize the identification of such pathogenic fungi, the mycobiota of the decaying stages of the unattended fungus comb were also assessed. The simultaneous assessment of the microbiota and the mycobiota of these stages identified possible interactions between the fungal and bacterial members of this community. Based on these, we propose a possible interaction among the crop fungusTermitomyces, the weedy fungusPseudoxylariaand some bacterial mutualists. These possibilities were then tested within vitrointeraction assays which suggest thatTermitomyces,Pseudoxylariaand bacterial mutualists all possess anti-fungal capabilities. We propose a multifactorial interaction model of these microbes, under the care of the termites, to explain how their interactions can maintain a predominantlyTermitomycesmonoculture.
Platelet Factor 4 (PF4) Improves Survival in a Murine Model of Antibiotic-Susceptible and Methicillin-ResistantStaphylococcus AureusPeritonitis
Authors: Podolnikova Nataly P. , Lishko Valeryi K. , Roberson Robert , Koh Zhqian , Derkach Dmitry , Richardson David , Sheller Michael , Ugarova Tatiana P.
Published: August 2023

The complement receptor CR3, also known as integrin Mac-1 (CD11b/CD18), is one of the major phagocytic receptors on the surface of neutrophils and macrophages. We previously demonstrated that in its protein ligands, Mac-1 binds sequences enriched in basic and hydrophobic residues and strongly disfavors negatively charged sequences. The avoidance by Mac-1 of negatively charged surfaces suggests that the bacterial wall and bacterial capsule possessing net negative electrostatic charge may repel Mac-1 and that the cationic Mac-1 ligands can overcome this evasion by acting as opsonins. Indeed, we previously showed that opsonization of Gram-negativeEscherichia coliwith several cationic peptides, including PF4 (Platelet Factor 4), strongly augmented phagocytosis by macrophages. Here, we investigated the effect of recombinant PF4 (rPF4) on phagocytosis of Gram-positiveStaphylococcus aureusin vitro and examined its impact in a mouse model ofS. aureusperitonitis. Characterization of the interaction of rPF4 with nonencapsulated and encapsulatedS. aureusshowed that rPF4 localizes on the bacterial surface, thus making it available for Mac-1. Furthermore, rPF4 did not have direct bactericidal and bacteriostatic activity and was not toxic to host cells. rPF4 enhanced phagocytosis ofS. aureusbioparticles by various primary and cultured Mac-1-expressing leukocytes by several folds. It also increased phagocytosis of live nonencapsulated and encapsulated bacteria. Notably, the augmentation of phagocytosis by rPF4 did not compromise the intracellular killing ofS. aureusby macrophages. Using a murineS. aureusperitonitis model, we showed that treatment of infected mice with rPF4 caused a significant increase in the clearance of antibiotic-susceptibleS. aureusand its methicillin-resistant (MRSA) variant and markedly improved survival. These findings indicate that rPF4 binding to the bacterial surface circumvents its antiphagocytic properties, improving host defense against antibiotic-susceptible and antibiotic-resistant bacteria.