bluuebunny/crossref_metadata_2025 huggingface dataset:
Monorhinal and Birhinal Odor Processing in Humans: an fMRI investigation
Authors: Ekanayake Anupa , Yang Qing , Kanekar Sangam , Ahmed Biyar , McCaslin Silas , Kalra Deepak , Eslinger Paul , Karunanayaka Prasanna
Published: August 2023

The olfactory nerve, also known as cranial nerve I, is known to have exclusive ipsilateral projections to primary olfactory cortical structures. It is still unclear whether these projections also correspond to functional pathways of odor processing. In an olfactory functional magnetic resonance imaging (fMRI) study of twenty young healthy subjects with a normal sense of smell, we tested whether nostril specific stimulation with phenyl ethyl alcohol (PEA), a pure olfactory stimulant, asymmetrically activates primary or secondary olfactory-related brain structures such as primary olfactory cortex, entorhinal cortex, and orbitofrontal cortex. The results indicated that without a challenging olfactory task, passive (no sniffing) and active (with sniffing) nostril-specific PEA stimulation did not produce asymmetrical fMRI activation in olfactory cortical structures.
PoMeLo: a systematic computational approach to predicting metabolic loss in pathogen genomes
Authors: Glascock Abigail Leigh , Waltari Eric , Dudas Gytis , Wong Joan , Ahyong Vida
Published: August 2023

Background

Genome streamlining, the process by which genomes become smaller and encode fewer genes over time, is a common phenomenon among pathogenic bacteria. This reduction is driven by selection for faster replication and minimized energy expenditure in a nutrientrich environment. As pathogens evolve to become more reliant on the host, metabolic genes and resulting capabilities are lost in favor of siphoning metabolites from the host. Characterizing genome streamlining, gene loss, and pathway degradation can be useful in assessing pathogen metabolic dependency on host metabolism and identifying potential targets for host-directed therapeutics.

Results

PoMeLo (PredictorofMetabolicLoss) is a novel evolutionary genomics-guided computational approach for identifying metabolic gaps in the genomes of pathogenic bacteria. PoMeLo leverages a centralized public database of high-quality genomes and annotations and allows the user to compare an unlimited number of genomes across individual genes and pathways. PoMeLo runs locally using user-friendly prompts in a matter of minutes and generates tabular and visual outputs for users to compare predicted metabolic capacity between groups of bacteria and individual species. Each pathway is assigned a Predicted Metabolic Loss (PML) score to assess the magnitude of genome streamlining. Optionally, PoMeLo places results in an evolutionary context by including phylogenetic relationships in visual outputs. It can also initially compute phylogenetically-weighted mean genome sizes to identify genome streamlining events. Here we describe PoMeLo and demonstrate its use in identifying metabolic gaps in genomes of pathogenicTreponemaspecies.

Conclusions

PoMeLo represents an advance over existing methods for identifying metabolic gaps in genomic data, allowing comparison across large numbers of genomes and placing the resulting data in a phylogenetic context. PoMeLo is freely available for academic and nonacademic use athttps://github.com/czbiohub-sf/pomelo.
SCO-spondin knockout mice exhibit small brain ventricles and mild spine deformation
Authors: Xu Huixin , Dugué Guillaume P. , Cantaut-Belarif Yasmine , Lejeune François-Xavier , Gupta Suhasini , Wyart Claire , Lehtinen Maria K.
Published: August 2023

Reissner’s fiber (RF) is an extracellular polymer comprising the large monomeric protein SCO-spondin (SSPO) secreted by the subcommissural organ (SCO) that extends through cerebrospinal fluid (CSF)-filled ventricles into the central canal of the spinal cord. In zebrafish, RF and CSF-contacting neurons (CSF-cNs) form an axial sensory system that detects spinal curvature, instructs morphogenesis of the body axis, and enables proper alignment of the spine. In mammalian models, RF has been implicated in CSF circulation. However, challenges in manipulatingSspo, an exceptionally large gene of 15,719 nucleotides, with traditional approaches has limited progress. Here, we generated aSspoknockout mouse model using CRISPR/Cas9-mediated genome-editing.Sspoknockout mice lacked RF-positive material in the SCO and fibrillar condensates in the brain ventricles. Remarkably,Sspoknockout brain ventricle sizes were reduced compared to littermate controls. Minor defects in thoracic spine curvature were detected inSspoknockouts, which did not alter basic motor behaviors tested. Altogether, our work in mouse demonstrates that SSPO and RF regulate ventricle size during development but only moderately impact spine geometry.
CDDO-Methyl Ester Inhibits BRAF Inhibitor Resistance and Remodels the Myeloid Compartment in BRAF-mutant Melanoma
Authors: Torres Gretel M. , Jarnagin Helen C. , Park Chanhyuk , Yang Heetaek , Kosarek Noelle N. , Bhandari Rajan , Wang Chen-Yu , Kolling Fred W. , Whitfield Michael L. , Turk Mary Jo , Liby Karen T. , Pioli Patricia A.
Published: August 2023

Approximately 50% of advanced melanomas harbor activating BRAFV600Emutations that are sensitive to BRAF inhibition. However, the duration of the response to BRAF inhibitors (BRAFi) has been limited due to the development of acquired resistance, which is preceded by recruitment of immunosuppressive myeloid cells and regulatory T cells (Tregs). While the addition of MAPK/ERK kinase 1 inhibitors (MEKi) prolongs therapeutic response to BRAF inhibition, most patients still develop resistance. Using a BrafV600E/+/Pten-/-graft mouse model of melanoma, we now show that the addition of the methyl ester of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (C-Me) to the BRAFi vemurafenib analog PLX4720 at resistance significantly reduces tumor burden. Dual treatment remodels the BRAFi resistant-tumor microenvironment (TME), reducing infiltration of Tregsand tumor associated macrophages (TAMs), and attenuates immunosuppressive cytokine production. For the first time, we characterize myeloid populations using scRNA-seq in BRAFi-resistant tumors and demonstrate that restoration of therapeutic response is associated with significant changes in immune-activated myeloid subset representation. Collectively, these studies suggest that C-Me inhibits acquired resistance to BRAFi. Use of C-Me in combination with other therapies may both inhibit melanoma growth and enhance therapeutic responsiveness more broadly.
Quantifying landscape-flux via single-cell transcriptomics uncovers the underlying mechanism of cell cycle
Authors: Zhu Ligang , Wang Jin
Published: August 2023

Recent developments in single-cell sequencing technology enable the acquisition of the whole transcriptome data. However, understanding the underlying mechanism and identifying the driving force of the transcriptional regulation of the cell function directly from these data remains challenging. To address this urgent need, we reconstruct a continuous vector field of cell cycle based on the discrete single-cell RNA velocity to quantify the single-cell global non-equilibrium dynamic landscape-flux. We reveal that large fluctuations disrupt the global landscape and genetic perturbations alter landscape-flux, thus identifying key genes in maintaining cell cycle dynamics and predicting associated effects on function. Additionally, we quantify the fundamental energy cost of the cell cycle initiation and reveal that sustaining the cell cycle requires curl flux and dissipation to maintain the oscillatory phase coherence. We enable the inference of the cell cycle gene regulatory networks directly from the single-cell transcriptomic data, including the feedback mechanisms and interaction intensity. This provides a golden opportunity to experimentally verify the landscape-flux theory and also obtain its associated quantifications. Our study also offers a unique framework for combining the landscape-flux theory and single-cell high-through sequencing experiments together for understanding the underlying mechanisms of the cell cycle and can be extended to other non-equilibrium biological processes, such as differentiation-development and disease pathogenesis.
Metabolic slowdown as the proximal cause of ageing and death
Authors: Wordsworth J. , Yde Nielsen P. , Fielder E. , Chandrasegaran S. , Shanley D.
Published: August 2023

Ageing results from the gradual loss of homeostasis, and there are currently many hypotheses for the underlying initial causes, such as molecular damage accumulation. However, few if any theories directly connect comprehensive, underlying biological mechanisms to specific age-related diseases. We recently demonstrated how a specific maintenance system impeding overactivity disorders such as cancer might undergo positive selection while still resulting in a gradual homeostatic shift toward slower metabolism. Here we connect this metabolic slowdown, via a series of unavoidable homeostatic shifts, to the hallmarks of ageing, including mitochondrial dysfunction, insulin resistance (IR), weight gain, basal inflammation, and age-related diseases such as atherosclerosis. We constructed the fuel and energy model (FEM) based on these shifts and found that ageing via metabolic slowdown could explain not only the effects of anti-ageing interventions such as rapamycin and calorie restriction, but many of the paradoxes of ageing that currently defy alternative theories.
Phenological mismatch between trees and wildflowers: Reconciling divergent findings in two recent analyses
Authors: Lee Benjamin R. , Alecrim Evelyn F. , Forrest Jessica R.K. , Heberling J. Mason , Primack Richard B. , Sargent Risa D.
Published: August 2023

Recent evidence suggests that community science and herbarium datasets yield similar estimates of species’ phenological sensitivities to temperature. Despite this, two recent studies by Alecrim et al. (2023) and Miller et al. (2022) found contradictory results when investigating an identical ecological mechanism (phenological mismatch of wildflower flowering and of shading by deciduous trees; “phenological escape”) with separate datasets.

Here, we investigated whether differences between the two studies’ results could be reconciled by testing four hypotheses related to model design, species selection, spatiotemporal data extent, and phenophase selection.

Hybrid model structures brought results from the two datasets closer together but did not fully reconcile the differences between the studies. Cropping the datasets to match spatial and temporal extents appeared to reconcile most differences but only at the cost of much higher uncertainty associated with reduced sample size. Neither species selection nor phenophase selection seemed to be responsible for differences in results.

Synthesis:Our analysis suggests that although species-level estimates of phenological sensitivity may be similar between crowd-sourced and herbarium datasets, inherent differences in the types and extent of data may lead to contradictory inference about complex biotic interactions. We conclude that, until community science data repositories grow to match the range of climate conditions present in herbarium collections or until herbarium collections grow to match the spatial extent and temporal frequency of community science repositories, ecological studies should ideally be evaluated using both datasets to test the possibility of biased results from either.
Antibody gene features associated with binding and functional activity in vaccine-derived human mAbs targeting malaria parasites
Authors: Coelho Camila H. , Marquez Susanna , Nguemwo Tentokam Bergeline C. , Berhe Anne D. , Miura Kazutoyo , Long Carole A. , Sagara Issaka , Healy Sara , Kleinstein Steven H. , Duffy Patrick E.
Published: August 2023

Adjuvants have been essential to malaria vaccine development, but their impact on the vaccine-induced antibody repertoire is poorly understood. Here, we used cDNA sequences from antigen-specific single memory B cells to express 132 recombinant human anti-Pfs230 monoclonal antibodies (mAbs). Alhydrogel®-induced mAbs demonstrated higher binding to Pfs230D1, although functional activity was similar between adjuvants. All Alhydrogel® mAbs using IGHV1-69 gene bound to recombinant Pfs230D1, but none blocked parasite transmission to mosquitoes; similarly, no AS01 mAb using IGHV1-69 blocked transmission. Functional mAbs from both Alhydrogel® and AS01 vaccines used IGHV3-21 and IGHV3-30 genes. Antibodies with the longest CDR3 sequences were associated with binding but not functional activity. This study assesses adjuvant effects on antibody clonotype diversity during malaria vaccination.
Does late water deficit induce root growth or senescence in wheat?
Authors: Shazadi Kanwal , Christopher John T. , Chenu Karine
Published: August 2023

Drought frequently limits productivity in rain-fed systems. To investigate water-stress impacts on post-anthesis root development in wheat, three experiments were conducted with two cultivars, Scout and Mace, grown in 1.5m tubes under well-watered conditions or post-anthesis water-stress. Shallow roots of both genotypes appeared to senesce between heading and maturity under well-watered conditions, whereas moderate water stress tended to increase post-heading shallow-root senescence in Mace but stimulated growth in Scout. For deep roots, net growth in biomass was observed for both genotypes under well-watered conditions between heading and maturity, whereas under moderate water stress, only Scout maintained net growth, with net senescence being observed for Mace. Severe water stress resulted in root senescence at all depths for both genotypes. Above ground, Scout retained leaf greenness for only slightly longer than Mace under well-watered conditions. In contrast, under moderate water stress, Mace showed rapid post-anthesis leaf senescence while Scout was affected little if at all. Grain biomass per plant was similar between genotypes in well-watered conditions but more reduced for Mace under moderate stress. Post-anthesis differences in root growth and senescence can strongly influence water use and grain filling in drought-prone environments. Screening for this could assist breeding for drought tolerant varieties.
Size dependent steady-state saturation limit in biomolecular transport through nuclear membranes
Authors: Shakhi P.K. , Bijeesh M.M. , Hareesh J. , Joseph Toby , Nandakumar P. , Varier Geetha K.
Published: August 2023

The nucleus preserves the genomic DNA of eukaryotic organisms and maintains the integrity of the cell by regulating the transport of molecules across the nuclear membrane. It is hitherto assumed that small molecules having a size below the passive permeability limit are allowed to diffuse freely to the nucleus while the transport of larger molecules are regulated via an active mechanism involving energy. Here we report on the kinetics of nuclear import and export of a model system of dextran molecules having a size below the passive permeability limit. The studies carried out using time-lapse confocal fluorescence microscopy show a clear deviation from the passive diffusion model. In particular, it is observed that the steady-state concentration of dextran molecules inside the nucleus is consistently less than the concentration outside, in contradiction to the predictions of the passive diffusion model. Detailed analysis and modeling of the transport show that the nuclear export rates significantly differ from the import rates and the difference in rates is dependent on the size of the molecules. The nuclear export rates are further confirmed by an independent experimental study where we observe the diffusion of dextran molecules from the nucleus directly. Our experiments and transport model would suggest that the nucleus actively rejects exogenous macromolecules even below the passive permeability limit. This result can have significant impact on biomedical research especially in areas related to targeted drug delivery and gene therapy.
Optimal antimicrobial response to a changing microbial background at a mucus interface
Authors: Bossa Guilherme Volpe , Bel Shai , Mugler Andrew , Erez Amir
Published: August 2023

Complex lifeforms host microbiota, microbes that live synergistically with their host. Accordingly, hosts have mechanisms to defend against and tolerate the microbiota. The intestinal mucus, where these systems collide, plays a pivotal role in managing this relationship, yet lacks an integrative theoretical framework. We propose a minimal model to elucidate dynamics at this interface, focusing on the ileum’s mucus defense. The model considers the effect of delay in host antimicrobial peptide secretion and how the host can use two different signals, from the bulk microbiota and from segmented filamentous bacteria (SFB). Our theory suggests the host can optimize defense by minimizing antimicrobial peptide production and controlling bacterial exposure. Integrating two recent experiments, we show host dynamics are consistent with sensing both bulk and SFB, supporting our ‘optimal defense’ hypothesis. Therefore, we propose that similar mechanisms could prove advantageous to other species and applicable beyond the ileum’s mucus barrier.
Dynamic instability of force-generating bacterial microtubules
Authors: Amini H. Reza , Volkov Vladimir A. , Dogterom Marileen
Published: August 2023

Dynamic instability refers to the ability of cytoskeletal polymers to switch between growing and shrinking phases. This phenomenon has been extensively studied for eukaryotic microtubules which consist of 13 protofilaments. Here we report on the dynamic properties of prokaryotic microtubules found inProsthecobacterbacteria, which consist of 4-5 protofilaments and, like their eukaryotic counterparts, display dynamic instability. Using microfabricated barriers we show that the catastrophe rate of bacterial microtubules increases when their growth is stalled by a rigid barrier. We find that the lifetime distributions of both free and stalled bacterial microtubules can be fitted using the same phenomenological model that we previously introduced for eukaryotic microtubules, suggesting that bacterial microtubules may be considered a model system for eukaryotic microtubules. We further use cryo-electron tomography to reveal structural details of dynamic ends and show that bacterial microtubules may form doublets similar to axonemal microtubules in eukaryotes.
Molecular Studies of Phages-Klebsiella pneumoniaein a Mucoid Environment: Innovative use of mucolytic agents prior to the administration of lytic phages
Authors: Pacios Olga , Blasco Lucía , Ortiz-Cartagena Concha , Bleriot Inés , Fernández-García Laura , López María , Barrio-Pujante Antonio , Pascual Álvaro , Cuenca Felipe Fernández , Martínez-Martínez Luis , Aracil Belén , Oteo-Iglesias Jesús , Tomás María
Published: August 2023

Mucins are important glycoproteins that form a protective layer throughout the gastrointestinal and respiratory tracts. There is scientific evidence of increase in phage-resistance in the presence of mucin for some bacterial pathogens. Manipulation in mucin composition may ultimately influence the effectiveness of phage therapy. In this work, two clinical strains ofK. pneumoniae(K3574 and K3325), were exposed to the lytic bacteriophage vB_KpnS-VAC35 in the presence and absence of mucin on a long-term co-evolution assay, in an attempt to mimicin vitrothe exposure to mucins that bacteria and their phages facein vivo. Enumerations of the bacterial and phage counts at regular time intervals were conducted, and extraction of the genomic DNA of co- evolved bacteria to the phage, the mucin and both was performed. We determined the frequency of phage-resistant mutants in the presence and absence of mucin and including a mucolytic agent (N-acetyl L-cysteine, NAC), and sequenced these conditions using Nanopore. We phenotypically demonstrated that the presence of mucin induces the emergence of bacterial resistance against lytic phages, effectively decreased in the presence of NAC. In addition, the genomic analysis revealed some of the genes relevant to the development of phage resistance in long-term co- evolution, with a special focus on the mucoid environment. Genes involved in the metabolism of carbohydrates were mutated in the presence of mucin. In conclusion, the use of mucolytic agents prior to the administration of lytic phages could be an interesting therapeutic option when addressingK. pneumoniaeinfections in environments where mucin is overproduced.
Dissecting Mechanisms of Ligand Binding and Conformational Changes in the Glutamine-Binding Protein
Authors: Han Zhongying , Panhans Sabrina , Brameyer Sophie , Bilgen Ecenaz , Ram Marija , Herr Anna , Narducci Alessandra , Isselstein Michael , Harris Paul D. , Brix Oliver , Jung Kirsten , Lamb Don C. , Lerner Eitan , Griffith Douglas , Weikl Thomas R. , Zijlstra Niels , Cordes Thorben
Published: August 2023

Ligand binding and conformational changes of biomacromolecules play a central role in the regulation of cellular processes. It is important to understand how both are coupled and what their role is in biological function. The biochemical properties, conformational states, and structural dynamics of periplasmic substrate-binding proteins (abbreviated SBPs or PBPs), which are associated with a wide range of membrane proteins, have been extensively studied over the past decades. Their ligand-binding mechanism, i.e., the temporal order of ligand-protein interactions and conformational changes, however, remains a subject of controversial discussion. We here present a biochemical and biophysical analysis of theE. coliglutamine-binding protein GlnBP concerning ligand binding and its coupling to conformational changes. For this, we used a combination of experimental techniques including isothermal titration calorimetry, single-molecule Förster resonance energy transfer, and surface-plasmon resonance spectroscopy. We found that both apo- and holo-GlnBP show no detectable exchange between open and (semi-)closed conformations on timescales between 100 ns and 10 ms. Furthermore, we also demonstrate that ligand binding and conformational changes in GlnBP are highly correlated. A global analysis of our results is consistent with a dominant induced-fit mechanism, where the ligand binds GlnBP prior to conformational rearrangements. Importantly, we suggest that the rigorous experimental and theoretical framework used here can be applied to other protein systems where the coupling mechanism of conformational changes and ligand binding is yet unclear or where doubts prevail.
Endocytosis gated by emergent properties of membrane-clathrin interactions
Authors: Wang Xinxin , Li Yueping , Liu Ailing , Padilla Ronnin , Lee Donghoon M. , Kim Daehwan , Mettlen Marcel , Chen Zhiming , Schmid Sandra L. , Danuser Gaudenz
Published: August 2023

Clathrin-mediated endocytosis (CME), the major cellular entry pathway, starts when clathrin assembles on the plasma membrane into clathrin-coated pits (CCPs). Two populations of CCPs are detected within the same cell: ‘productive’ CCPs that invaginate and pinch off, forming clathrin-coated vesicles (CCVs) [1, 2], and ‘abortive’ CCPs [3, 4, 5] that prematurely disassemble. The mechanisms of gating between these two populations and their relations to the functions of dozens of early-acting endocytic accessory proteins (EAPs) [5, 6, 7, 8, 9] have remained elusive. Here, we use experimentally-guided modeling to integrate the clathrin machinery and membrane mechanics in a single dynamical system. We show that the split between the two populations is an emergent property of this system, in which a switch between anOpenstate and aClosedstate follows from the competition between the chemical energy of the clathrin basket and the mechanical energy of membrane bending.In silicoexperiments revealed an abrupt transition between the two states that acutely depends on the strength of the clathrin basket. This critical strength is lowered by membrane-bending EAPs [10, 11, 12]. Thus, CME is poised to be shifted between abortive and productive events by small changes in membrane curvature and/or coat stability. This model clarifies the workings of a putative endocytic checkpoint whose existence was previously proposed based on statistical analyses of the lifetime distributions of CCPs [4, 13]. Overall, a mechanistic framework is established to elucidate the diverse and redundant functions of EAPs in regulating CME progression.
Trichinella spiralis secretes stage-specific extracellular vesicles for modulating host microenvironment for parasitism
Authors: Khueangchiangkhwang Sukhonthip , Wu Zhiliang , Hosoda-Yabe Ritsuko , Yabe Tomio , Okamoto Toru , Takada Chihiro , Okada Hideshi , Ogura Shinji , Maekawa Yoichi
Published: August 2023

Extracellular vesicles (EVs) regulate cell-to-cell communication by transferring biomolecules within individuals or between different organisms. Trichinella spiralis is thought to secrete EVs to establish parasitism, but the detailed mechanisms of EV-mediated parasitic strategies have remained unclear. We identified EVs of T. spiralis as stage-specific in adult worms in host intestine (AW-EVs) and larvae in host muscles (ML-EVs). Stage-specific EVs exhibited unique functions, such as suppressing the expression of mucin-related genes only in AW-EVs and inducing genes associated with myoblast differentiation found in nurse cell formation in ML-EVs. Both EVs also suppressed IL-6 expression. These functions corelated with the contents of each EV, as confirmed by proteomics and whole microRNA sequencing. One of the microRNAs common to both EVs, tsp-miR-1, was involved in the suppression of IL-6 expression. These results suggest that Trichinella spiralis secretes stage-specific EVs containing unique as well as common biomolecules to regulate the host microenvironment for parasitism.
Heterologous expression of influenza hemagglutinin leads to early and transient activation of the unfolded protein response inNicotiana benthamiana
Authors: Hamel Louis-Philippe , Comeau Marc-André , Tardif Rachel , Gravel Francis Poirier- , Paré Marie-Ève , Lavoie Pierre-Olivier , Goulet Marie-Claire , Michaud Dominique , D’Aoust Marc-André
Published: August 2023

The unfolded protein response (UPR) allows cells to cope with endoplasmic reticulum (ER) stress induced by the accumulation of misfolded proteins in the ER. Due to its sensitivity toAgrobacterium tumefaciens, model plantNicotiana benthamianais widely employed for the transient expression of recombinant proteins of biopharmaceutical interest, including therapeutic antibodies and virus surface proteins used for vaccine production. As such, study of the plant UPR is of practical significance, since enforced expression of complex secreted proteins often results in ER stress. After 6 days of expression, we recently reported that influenza hemagglutinin (HA) induces accumulation of UPR proteins. Since the upregulation of corresponding UPR genes was not detected at this time point, accumulation of UPR proteins was hypothesized to either be independent of transcriptional regulation, or associated with early but transient UPR gene upregulation. Using time course sampling, we here show that HA expression does result in early and transient activation of the UPR, as inferred from unconventional splicing ofNbbZIP60transcripts and induction of UPR genes with varied functions. The transient nature of HA-induced UPR suggests that this response was sufficient to cope with ER stress provoked by expression of the secreted protein, as opposed to an antibody that triggered a stronger and more sustained UPR. As defense-related genes were induced after the peak of UPR activation and correlated with high increase in HA protein accumulation, we hypothesize that these immune responses, rather than the UPR, were responsible for the onset of necrotic symptoms on HA-expressing leaves.

One-sentence summary

Agrobacterium-mediated expression of influenza hemagglutinin results in early and transient activation of the unfolded protein response, preventing deleterious effects caused by unresolved endoplasmic reticulum stress.
A Phosphoproteomics Data Resource for Systems-level Modeling of Kinase Signaling Networks
Authors: Feng Song , Sanford James A. , Weber Thomas , Hutchinson-Bunch Chelsea M. , Dakup Panshak P. , Paurus Vanessa L. , Attah Kwame , Sauro Herbert M. , Qian Wei-Jun , Wiley H. Steven
Published: August 2023

Building mechanistic models of kinase-driven signaling pathways requires quantitative measurements of protein phosphorylation across physiologically relevant conditions, but this is rarely done because of the insensitivity of traditional technologies. By using a multiplexed deep phosphoproteome profiling workflow, we were able to generate a deep phosphoproteomics dataset of the EGFR-MAPK pathway in non-transformed MCF10A cells across physiological ligand concentrations with a time resolution of <12 min and in the presence and absence of multiple kinase inhibitors. An improved phosphosite mapping technique allowed us to reliably identify >46,000 phosphorylation sites on >6600 proteins, of which >4500 sites from 2110 proteins displayed a >2-fold increase in phosphorylation in response to EGF. This data was then placed into a cellular context by linking it to 15 previously published protein databases. We found that our results were consistent with much, but not all previously reported data regarding the activation and negative feedback phosphorylation of core EGFR-ERK pathway proteins. We also found that EGFR signaling is biphasic with substrates downstream of RAS/MAPK activation showing a maximum response at <3ng/ml EGF while direct substrates, such as HGS and STAT5B, showing no saturation. We found that RAS activation is mediated by at least 3 parallel pathways, two of which depend on PTPN11. There appears to be an approximately 4-minute delay in pathway activation at the step between RAS and RAF, but subsequent pathway phosphorylation was extremely rapid. Approximately 80 proteins showed a >2-fold increase in phosphorylation across all experiments and these proteins had a significantly higher median number of phosphorylation sites (~18) relative to total cellular phosphoproteins (~4). Over 60% of EGF-stimulated phosphoproteins were downstream of MAPK and included mediators of cellular processes such as gene transcription, transport, signal transduction and cytoskeletal arrangement. Their phosphorylation was either linear with respect to MAPK activation or biphasic, corresponding to the biphasic signaling seen at the level of the EGFR. This deep, integrated phosphoproteomics data resource should be useful in building mechanistic models of EGFR and MAPK signaling and for understanding how downstream responses are regulated.
Migrasome formation is initiated preferentially in tubular junctions by alternations of membrane tension or intracellular pressure
Authors: Zucker Ben , Dharan Raviv , Wang Dongju , Yu Li , Sorkin Raya , Kozlov Michael M.
Published: August 2023

Migrasomes, the transient vesicle-like cellular organelles, arise on the retraction fibers (RFs), the branched tubular extensions of the plasma membrane generated during cell migration. Migrasomes form in two steps: a local RF swelling is followed by a protein-dependent stabilization of the emerging spherical bulge. Here we approached experimentally and theoretically the previously unaddressed mechanism of the initial RF swelling. We hypothesized that the swelling can be driven by alterations of the generic mechanical factors, the RF’s luminal pressure and membrane tension. To examine the effects of pressure, we exposed migrating RF-producing cells to a hypotonic medium and observed the formation of migrasome-like bulges with a preferential location in the RF branching sites. To test the results of tension variations, we developed a biomimetic system of three membrane tubules connected by a junction and subjected to controlled membrane tension. An abrupt increase of tension resulted in a migrasome-like bulge formation in the junction and in the tubular regions. Following the formation, the tubule’s bulges moved toward and merged with the junctional bulge. To understand the physical forces behind the observations, we considered theoretically the mechanical energy of a membrane system consisting of a three-way tubular junction with emerging tubular arms connected to a membrane reservoir. The energy minimization predicted the membrane bulging, preferably, in the junction site as a result of both an increase in the luminal pressure and an abrupt rise of the membrane tension. We discuss the common physical background of the two phenomena.
ATAC and SAGA coactivator complexes utilize co-translational assembly, but their cellular localization properties and functions are distinct
Authors: Yayli Gizem , Bernardini Andrea , Mendoza Sanchez Paulina Karen , Scheer Elisabeth , Damilot Mylène , Essabri Karim , Morlet Bastien , Negroni Luc , Vincent Stéphane D. , Timmers HT Marc , Tora László
Published: August 2023

To understand the function of multisubunit complexes it is of key importance to uncover the precise mechanisms that guide their assembly. Nascent proteins can find and bind their interaction partners during their translation, leading to co-translational assembly. Here we demonstrate that the core modules of ATAC (ADA-Two-A-Containing) and SAGA (Spt-Ada-Gcn5-acetyltransferase), two lysine acetyl transferase-containing transcription coactivator complexes, assemble co-translationally in the cytoplasm of mammalian cells. In addition, SAGA complex containing all of its modules forms in the cytoplasm and acetylates non-histones proteins. In contrast, fully assembled ATAC complex cannot be detected in the cytoplasm of mammalian cells. However, endogenous ATAC complex containing two functional modules forms and functions in the nucleus. Thus, the two related coactivators, ATAC and SAGA, assemble by using co-translational pathways, but their subcellular localization, cytoplasmic abundance and functions are distinct.
Evaluation of Fendiline Treatment in VP40 System with Nucleation-Elongation Process: A Computational Model of Ebola Virus Matrix Protein Assembly
Authors: Liu Xiao , Husby Monica , Stahelin Robert V. , Pienaar Elsje
Published: August 2023

Ebola virus (EBOV) infection is threatening human health, especially in Central and West Africa. Limited clinical trials and the requirement of biosafety level-4 (BSL-4) laboratories hinders experimental work to advance our understanding of EBOV and evaluation of treatment. In this work, we use a computational model to study the assembly and budding process of EBOV and evaluate the effect of fendiline on these processes. Our results indicate that the assembly of VP40 filaments may follow the nucleation-elongation theory, as it is critical to maintain a pool of VP40 dimer for the maturation and production of virus-like particles (VLPs). We further find that the nucleation-elongation process can also be influenced by phosphatidylserine (PS), which can complicate the efficacy of fendiline, a drug that lowers cellular PS levels. We observe that fendiline may increase VLP production at earlier time points (24 h) and under low concentrations (≤ 2 μM). But this effect is transient and does not change the conclusion that fendiline generally decreases VLP production. We also conclude that fendiline can be more efficient at the stage of VLP budding relative to earlier phases. Combination therapy with a VLP budding step-targeted drug may further increase the treatment efficiency of fendiline. Finally, we also show that fendiline has higher efficacy when VP40 expression is high. While these are single-cell level results based on the VP40 system, it points out a potential way of fendiline application affecting EBOV assembly, which can be further tested in experimental studies with multiple EBOV proteins or live virus.

Importance

EBOV infection can cause deadly hemorrhagic fever, which has a mortality rate around 90% without treatment. The recent outbreaks in Uganda and Democratic Republic of the Congo illustrate its treat to human health. Though two antibody-based treatments are approved, mortality rates in the last outbreak is still higher than 30%. This can partly be due to the requirement of advanced medical facilities for current treatments. As a result, it is very important to develop and evaluate new therapies for EBOV infection, especially those can be easily applied in the developing world. The significance of our research is that we evaluate the potential treatment effect of fendiline on EBOV infection in the VP40 system with a computational approach, which both greatly saves time and lowers cost compared to traditional experimental studies, and provides innovative new tools to study viral protein dynamics.
The Prp19C subunits Cwc15 and Syf2 function in TREX occupancy and transcription elongation
Authors: Henke-Schulz Laura , Minocha Rashmi , Sträßer Katja
Published: August 2023

The Prp19 complex (Prp19C) is conserved from yeast to human and functions in many different processes such as genome stability, splicing and transcription elongation. In the latter, Prp19C ensures TREX occupancy at transcribed genes. TREX in turn couples transcription to nuclear mRNA export by recruiting the mRNA exporter to transcribed genes and consequently to nascent mRNAs. Here, we assess the function of the nonessential Prp19C subunits Syf2 and Cwc15 in the interaction of Prp19C and TREX with the transcription machinery, Prp19C and TREX occupancy as well as transcription elongation. Whereas both proteins are important for Prp19C-TREX interaction, Syf2 is needed for full Prp19C occupancy, and Cwc15 is important for the interaction of Prp19C with RNA polymerase II and TREX occupancy. These partially overlapping functions are corroborated by a genetic interaction betweenΔcwc15andΔsyf2. Finally, Cwc15 also interacts genetically with the transcription elongation factor Dst1 and functions in transcription elongation. In summary, we uncover novel roles of the nonessential Prp19C components Syf2 and Cwc15 in Prp19C’s function in transcription elongation.
Structure of the completeS. cerevisiaeRpd3S-nucleosome complex
Authors: Markert Jonathan W. , Vos Seychelle M. , Farnung Lucas
Published: August 2023

Acetylation of histones is a key post-translational modification that guides gene expression regulation. In yeast, the class I histone deacetylase containing Rpd3S complex plays a critical role in the suppression of spurious transcription by removing histone acetylation from actively transcribed genes. TheSaccharomyces cerevisiaeRpd3S complex has five subunits (Rpd3, Sin3, Rco1, Eaf3, and Ume1) but its subunit stoichiometry and how the complex engages nucleosomes to achieve substrate specificity remains elusive. Here we report the cryo-EM structure of the complete Rpd3S complex bound to a nucleosome. Sin3 and two copies of subunits Rco1 and Eaf3 encircle the deacetylase subunit Rpd3 and coordinate the binding of Ume1. The Rpd3S complex binds both trimethylated H3 tails at position lysine 36 and makes multiple additional contacts with the nucleo-somal DNA, the H2A–H2B acidic patch, and histone H3. Direct regulation via the Sin3 subunit coordinates binding of the acetylated histone substrate to achieve substrate specificity.
A unifying computational account of temporal context effects in language across the human cortex
Authors: Vo Vy Ai , Jain Shailee , Beckage Nicole , Chien Hsiang-Yun Sherry , Obinwa Chiadika , Huth Alexander G.
Published: August 2023

Deep learning advances have revolutionized computational modeling approaches in neuroscience. However, their black-box nature makes it challenging to use deep learning models to discover new insights about brain function. Focusing on human language processing, we propose a new framework to improve the quality and interpretability of the inferences we make from deep learning-based models. First, we add interpretable components to a deep language model and use it to build a predictive encoding model. Then, we use the model’s predictive abilities to simulate brain responses to controlled stimuli from published experiments. We find that our model, based on a multi-timescale recurrent neural network, captures many previously reported temporal context effects in human cortex. Its failure to capture other effects also highlights important gaps in current language models. Finally, we use this new framework to generate model-based evidence that supports the proposal that different linguistic features are represented at different timescales across cortex.
Number of kinesins engaged in axonal cargo transport: A novel biomarker for neurological disorders
Authors: Hayashi Kumiko , Sasaki Kazuo
Published: August 2023

Kinesin motor proteins play crucial roles in anterograde transport of cargo vesicles in neurons, moving them along axons from the cell body towards the synaptic region. Not only the transport force and velocity of single motor protein, but also the number of kinesin molecules involved in transporting a specific cargo, is pivotal for synapse formation. This collective transport by multiple kinesins ensures stable and efficient cargo transport in neurons. Abnormal increases or decreases in the number of engaged kinesin molecules per cargo could potentially act as biomarkers for neurodegenerative diseases such as Alzheimer’s, Parkinson’s, amyotrophic lateral sclerosis (ALS), spastic paraplegia, polydactyly syndrome, and virus transport disorders. We review here a model constructed using physical measurements to quantify the number of kinesin molecules associated with their cargo, which could shed light on the molecular mechanisms of neurodegenerative diseases related to axonal transport.
The structure of immature tick-borne encephalitis virus
Authors: Anastasina Maria , Füzik Tibor , Domanska Aušra , Pulkkinen Lauri IA , Šmerdová Lenka , Formanová Petra Pokorná , Straková Petra , Nováček Jiří , Růžek Daniel , Plevka Pavel , Butcher Sarah J
Published: August 2023

Tick-borne encephalitis virus (TBEV) is a medically important flavivirus that poses a significant health threat in Europe and Asia. However, the structure of the immature form of TBEV remains unknown. Here, we employed state-of-the-art cryogenic electron microscopy (cryoEM) to determine the structure of the immature TBEV particle. The immature TBEV particle has a diameter of 56 nm and its surface glycoproteins are organised into spikes characteristic of immature flaviviruses. The cryoEM reconstructions of the whole virus and of the individual spike enabled us to build atomic models of the major viral components, the E and prM proteins. The insights obtained from our study provide a foundation for understanding the early stages of TBEV assembly and maturation. The pr domains of prM have a critical role in holding the heterohexameric prM3E3 spikes in metastable conformation. Destabilisation of the prM furin-sensitive loop at acidic pH facilitates its processing. The prM cleavage, the collapse of E protein ectodomains onto the virion surface concurrent with significant movement of the membrane domains of both E and M, and release of the pr fragment from the particle render the virus mature and infectious. This knowledge contributes to our understanding of the flavivirus life cycle.
Carbapenem- and cefiderocol-resistantEnterobacteralesin surface waters in Kumasi, Ashanti Region, Ghana
Authors: Eger Elias , Homeier-Bachmann Timo , Adade Eugene , Dreyer Sylvia , Heiden Stefan E. , Tawiah Patrick Ofori , Sylverken Augustina Angelina , Knauf Sascha , Schaufler Katharina
Published: August 2023

This study investigated the presence of extended-spectrum β-lactamase-producingEnterobacteralesin selected surface waters in the Ashanti Region in Ghana. Subsequent genomic analysis revealed that one-fifth of the isolates were genotypically carbapenem-resistant. Phenotypic susceptibility testing not only confirmed their carbapenem resistance, but also uncovered two cefiderocol-resistant isolates.
ArreSTick Motif is Responsible for GPCR-β-Arrestin Binding Stability and Extends Phosphorylation-Dependent β-arrestin Interactions to Non-Receptor Proteins
Authors: Tóth András Dávid , Soltész-Katona Eszter , Kis Katalin , Guti Viktor , Gilzer Sharon , Prokop Susanne , Boros Roxána , Misák Ádám , Balla András , Várnai Péter , Turiák Lilla , Ács András , Drahos László , Inoue Asuka , Hunyady László , Turu Gábor
Published: August 2023

The binding and function of β-arrestins are regulated by specific phosphorylation motifs present in G protein-coupled receptors (GPCRs). However, the exact arrangement of phosphorylated amino acids responsible for establishing a stable interaction remains unclear. To investigate this pattern, we employed a 1D sequence convolution model trained on a dataset of GPCRs that have established β-arrestin binding properties. This approach allowed us to identify the amino acid pattern required for GPCRs to form stable interactions with β-arrestins. This motif was named “arreSTick.” Our data show that the model predicts the strength of the coupling between GPCRs and β-arrestins with high accuracy, as well as the specific location of the interaction within the receptor sequence. Furthermore, we show that the arreSTick pattern is not limited to GPCRs, and is also present in numerous non-receptor proteins. Using a proximity biotinylation assay and mass spectrometry analysis, we demonstrate that the arreSTick motif controls the interaction between numerous non-receptor proteins and β-arrestins. For example, the HIV-1 Tat Specific Factor 1 (HTSF1 or HTATSF1), a nuclear transcription factor, contains the arreSTick pattern, and our data show that its subcellular localization is influenced by its coupling to β-arrestin2. Our findings unveil a broader regulatory role for β-arrestins in phosphorylation-dependent interactions, extending beyond GPCRs to encompass non-receptor proteins as well.
Performance and allocation can simultaneously shape behaviour in fire-disturbed populations of root vole
Authors: Boratyński Jan S. , Iwińska Karolina , Wirowska Martyna , Borowski Zbigniew , Solecki Paweł , Ciesielski Mariusz , Boratyński Zbyszek
Published: August 2023

Metabolic physiology and animal personality are often considered linked to each other, shaping ecological and evolutionary strategies along a life-history continuum. The energy allocation model predicts a negative while the performance model predicts a positive correlation between the rate of metabolic processes and behaviours, such as activity level. The models might operate simultaneously but depending on the context one can predominate over the other, determining expression of alternative pro- and reactive behavioural strategies. Large-scale fires, such as the one that burnt wetlands of Biebrza National Park (NE Poland), degrade natural habitats, affect amount of food and shelters and modify predatory-prey interactions. Fires pose also direct threat to survival of local populations, such as the wetland specialist root vole (Microtus oeconomus). We hypothesized that fire disturbance, by changing environmental context and selective regimes, determines mechanisms linking physiology and behaviour. Positive relation found among most studies, predicted by the performance model, would revert to negative relation, predicted by the allocation model, affecting animals ecological strategy in disturbed habitat. We repeatedly measured maintenance and exercise metabolic rates and activity behaviour on voles from post-fire and unburnt populations. Repeatable maintenance metabolism and activity level were positively correlated, but more labile exercise metabolism did not explain behaviour. The correlations were not strongly affected by fire disturbance, but voles from post-fire habitat had higher maintenance but not maximum metabolism and moved shorter distances than individuals from unburnt area. The results suggest that performance model predominates, while habitat disturbance might reveal some allocation constraints on physiology-personality linkage.

Summary statement

Contrasting ’allocation’ and ’performance’ models, for energetics-behaviour linkage, were tested in context of fire-disturbance. Positive (performance) correlation predominated but animals from burned habitat had elevated metabolism and suppressed exploration (allocation).