bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteomic Analysis of Dorsal Root Ganglia in a Mouse Model of Paclitaxel-Induced Neuropathic Pain
Authors: Hanna Rania , Graur Alexandru , Sinclair Patricia , Mckiver Bryan D. , Bos Paula D. , Damaj M. Imad , Kabbani Nadine
Published: June 2024

Paclitaxel is a chemotherapy drug widely used for the treatment of various cancers based on its ability to potently stabilize cellular microtubules and block division in cancer cells. Paclitaxel-based treatment, however, accumulates in peripheral system sensory neurons and leads to a high incidence rate (over 60%) of chemotherapy induced peripheral neuropathy. Using an established preclinical model of paclitaxel-induced peripheral neuropathy (PIPN), we examined proteomic changes in dorsal root ganglia (DRG) of adult male mice that were treated with paclitaxel (8 mg/kg, at 4 injections every other day) relative to vehicle-treated mice. High throughput proteomics based on liquid chromatography electrospray ionization mass spectrometry identified 165 significantly altered proteins in lumbar DRG. Gene ontology enrichment and bioinformatic analysis revealed an effect of paclitaxel on pathways for mitochondrial regulation, axonal function, and inflammatory purinergic signaling as well as microtubule activity. These findings provide insight into molecular mechanisms that can contribute to PIPN in patients.
Sex and Disease Regulate MHC I Expression in Human Lung Epithelial Cells
Authors: Mathé Justine , Brochu Sylvie , Adam Damien , Brochiero Emmanuelle , Perreault Claude
Published: June 2024

Major histocompatibility complex class I (MHC I) molecules present endogenous peptides to CD8+ T-cells for immunosurveillance of infections and cancers. Recent studies revealed unexpected heterogeneity in MHC I expression among cells of different lineages. While respiratory diseases rank among the leading causes of mortality, studies in mice showed that lung epithelial cells (LECs) express lower MHC I levels than all other tested cell types. The present study aimed to evaluate MHC I expression in human LECs from parenchymal explants using single-cell RNA sequencing (scRNA-seq) and immunostaining of primary human LECs. After confirming the low constitutive MHC I expression in human LECs, we observed a significant upregulation of MHC I across three chronic respiratory diseases: chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and cystic fibrosis (CF). Additionally, we unveiled an unexpected sexual dimorphism in MHC I expression in both health and disease, with males exhibiting higher levels of MHC I under steady-state conditions. Gene expression analyses suggest that differential redox balance between sexes is instrumental in this dimorphism. Our study unveils the complex interplay between MHC I expression, sex, and respiratory diseases. Since, in other models, MHC I upregulation contributes to the development of immunopathologies, we propose that it might have a similar impact on chronic lung diseases.

NEW & NOTEWORTHY

This study shows that MHC I expression is very low in healthy LECs but escalates significantly in three chronic respiratory diseases, potentially contributing to disease progression. Furthermore, sex-specific divergences in LEC MHC I levels hint at distinct susceptibilities to chronic lung inflammation between males and females.

Graphical abstract
TarDis: Achieving Robust and Structured Disentanglement of Multiple Covariates
Authors: Inecik Kemal , Kara Aleyna , Rose Antony , Haniffa Muzlifah , Theis Fabian J.
Published: June 2024

Addressing challenges in domain invariance within single-cell genomics necessitates innovative strategies to manage the heterogeneity of multi-source datasets while maintaining the integrity of biological signals. We introduceTarDis, a novel deep generative model designed to disentangle intricate covariate structures across diverse biological datasets, distinguishing technical artifacts from true biological variations. By employing tailored covariate-specific loss components and a self-supervised approach,TarDiseffectively generates multiple latent space representations that capture each continuous and categorical target covariate separately, along with unexplained variation. Our extensive evaluations demonstrate thatTarDisoutperforms existing methods in data integration, covariate disentanglement, and robust out-of-distribution predictions. The model’s capacity to produce interpretable and structured latent spaces, including ordered latent representations for continuous covariates, enhances its utility in hypothesis-driven research. Consequently,TarDisoffers a promising analytical platform for advancing scientific discovery, providing insights into cellular dynamics, and enabling targeted therapeutic interventions.
Suppressing phagocyte activation by overexpressing the phosphatidylserine lipase ABHD12 preserves sarmopathic nerves
Authors: Dingwall Caitlin B. , Sasaki Yo , Strickland Amy , Summers Daniel W. , Bloom A. Joseph , DiAntonio Aaron , Milbrandt Jeffrey
Published: June 2024

Programmed axon degeneration (AxD) is a key feature of many neurodegenerative diseases. In healthy axons, the axon survival factor NMNAT2 inhibits SARM1, the central executioner of AxD, preventing it from initiating the rapid local NAD+ depletion and metabolic catastrophe that precipitates axon destruction. Because these components of the AxD pathway act within neurons, it was also assumed that the timetable of AxD was set strictly by a cell-intrinsic mechanism independent of neuron-extrinsic processes later activated by axon fragmentation. However, using a rare human disease model of neuropathy caused by hypomorphic NMNAT2 mutations and chronic SARM1 activation (sarmopathy), we demonstrated that neuronal SARM1 can initiate macrophage-mediated axon elimination long before stressed-but-viable axons would otherwise succumb to cell-intrinsic metabolic failure. Investigating potential SARM1-dependent signals that mediate macrophage recognition and/or engulfment of stressed-but-viable axons, we found that chronic SARM1 activation triggers axonal blebbing and dysregulation of phosphatidylserine (PS), a potent phagocyte immunomodulatory molecule. Neuronal expression of the phosphatidylserine lipase ABDH12 suppresses nerve macrophage activation, preserves motor axon integrity, and rescues motor function in this chronic sarmopathy model. We conclude that PS dysregulation is an early SARM1-dependent axonal stress signal, and that blockade of phagocytic recognition and engulfment of stressed-but-viable axons could be an attractive therapeutic target for management of neurological disorders involving SARM1 activation.
Cell type-specific immune regulation under symbiosis in a facultatively symbiotic coral
Authors: Valadez-Ingersoll Maria , Rivera Hanny E. , Da-Anoy JK , Kanke Matthew R. , Gomez-Campo Kelly , Martinez-Rugerio M. Isabel , Kwan Julian , Hekman Ryan , Emili Andrew , Gilmore Thomas D. , Davies Sarah W.
Published: June 2024

Many cnidarians host single-celled algae within gastrodermal cells, yielding a mutually beneficial exchange of nutrients between host and symbiont, and dysbiosis can lead to host mortality. Previous research has uncovered symbiosis tradeoffs, including suppression of immune pathways in cnidarians hosting intracellular algae and correlations between symbiotic state and pathogen susceptibility. Here, we used a multiomic approach to characterize symbiotic states of the facultatively symbiotic coralOculina arbusculaby generating genotype-controlled fragments of symbiotic and aposymbiotic tissue. 16S metabarcoding showed no difference in bacterial communities between symbiotic states. Whole-organism proteomics revealed differential abundance of proteins related to immunity, confirming immune suppression during symbiosis. Finally, single-cell RNAseq identified diverse cell clusters within seven cell types across symbiotic states. Specifically, the gastrodermal cell clusters containing algal-hosting cells from symbiotic tissue had higher expression of nitrogen cycling and sugar transport genes than aposymbiotic gastrodermal cells. Furthermore, differential enrichment of immune system gene pathways and lower expression of genes involved in immune regulation were observed in these gastrodermal cells from symbiotic tissue. However, no differences in immune gene expression in the immune cell cluster were observed between symbiotic states. This work reveals a compartmentalization of immune system regulation in specific gastrodermal cells in symbiosis, which may limit symbiosis tradeoffs by simultaneously dampening immunity in algal-hosting cells while maintaining general organismal immunity.
Characterizing interactions in the nuclear pore complex transporter using novel site-specific deuteration and SANS
Authors: Tawani Arpita , Qian Shuo , Sparks Samuel E , Sashi Piakulla , Cahill Sean , Rout Michael , Cowburn David
Published: June 2024

We describe an unprecedented SANS/solvent matched experiment with the aim of providing a meso-scale dynamic structural description of fuzzy complexes such as those formed by nuclear transport factors as they ferry cargo across the nuclear pore complex, whose description is normally limited by the large number of states and rapid time scales of interconversion. These contain repeat short linear interaction domains which are common features of many intrinsically disordered functional proteins - e.g., transcriptional regulators, and RNA-interaction domains associated with liquid-liquid phase separated non-membranous organelles. The novel approach uses site-specific deuteration, SANS, and model fitting to provide changes to average spatial distributions between interacting domains of FG Nups upon binding to the nuclear transport factor NTF2. The results support the fully disordered nature of phenylalanyl-glycyl repeats within FG Nups in their interactions with the nuclear transport factor in vitro, as well as the absence of significant inter-aromatic contacts, or of interchain linkage in complexes.
Weakly nonlinear responses at low intrinsic noise levels in two types of electrosensory primary afferents
Authors: Barayeu Alexandra , Schlungbaum Maria , Lindner Benjamin , Benda Jan , Grewe Jan
Published: June 2024

Neuronal processing is inherently nonlinear — spiking thresholds or rectification in synapses are central to neuronal computations. Nevertheless, linear response theory has been instrumental in understanding, for example, the impact of noise or synchronous spikes on signal transmission, or the emergence of oscillatory activity. At higher signal-to-noise ratios, however, the third term in the Volterra series becomes relevant. This second-order susceptibility captures nonlinear interactions between pairs of stimulus frequencies. Theoretical results for leaky integrate-and-fire neurons suggest strong responses at the sum of two input frequencies only when these frequencies or their sum match the neuron’s baseline firing rate. We here analyze second-order susceptibilities in two types of primary electroreceptor afferents, P-units of the active and ampullary cells of the passive electrosensory system of the wave-type electric fishApteronotus leptorhynchus. In our combined experimental and modeling approach we find the predicted weakly nonlinear responses in some P-units with very low baseline interspike-interval variability and much stronger in all ampullary cells, which are less noisy than P-units. Such nonlinear responses boost responses to weak sinusoidal stimuli and are therefore of immediate relevance for wave-type electric fish that are exposed to superpositions of many frequencies in social contexts.
TransLeish: Identification of membrane transporters essential for survival of intracellularLeishmaniaparasites in a systematic gene deletion screen
Authors: Albuquerque-Wendt Andreia , McCoy Ciaran , Neish Rachel , Dobramysl Ulrich , Beneke Tom , Cowley Sally A. , Crouch Kathryn , Wheeler Richard J. , Mottram Jeremy C. , Gluenz Eva
Published: June 2024

For the protozoan parasiteLeishmania, completion of its life cycle requires sequential adaptation of cellular physiology and nutrient scavenging mechanisms to the different environments of a sand fly alimentary tract and the acidic mammalian host cell phagolysosome. Transmembrane transporters are the gatekeepers of intracellular environments, controlling the flux of solutes and ions across membranes. To discover which transporters are vital for survival as intracellular amastigote forms, we carried out a systematic loss-of-function screen of theL. mexicanatransportome. A total of 312 protein components of small molecule carriers, ion channels and pumps were identified and targeted in a CRISPR-Cas9 gene deletion screen in the promastigote form, yielding 188 viable null mutants. Forty transporter deletions caused significant loss of fitness in macrophage and mouse infections. A striking example is the Vacuolar H+ATPase (V-ATPase), which, unexpectedly, was dispensable for promastigote growthin vitrobut essential for survival of the disease-causing amastigotes.
Disordered regions of human eIF4B orchestrate a dynamic self-association landscape
Authors: Swain Bikash Chandra , Sarkis Pascale , Ung Vanessa , Rousseau Sabrina , Fernandez Laurent , Meltonyan Ani , Aho V. Esperance , Mercadante Davide , Mackereth Cameron D. , Aznauryan Mikayel
Published: June 2024

Eukaryotic translation initiation factor eIF4B is required for efficient cap-dependent translation, it is overexpressed in cancer cells, and may influence stress granule formation. Due to the high degree of intrinsic disorder, eIF4B is rarely observed in cryo-EM structures of translation complexes and only ever by its single structured RNA recognition motif domain, leaving the molecular details of its large intrinsically disordered region (IDR) unknown. By integrating experiments and simulations we demonstrate that eIF4B IDR orchestrates and fine-tunes an intricate transition from monomers to a condensed phase, in which large-size dynamic oligomers form before mesoscopic phase separation. Single-molecule spectroscopy combined with molecular simulations enabled us to characterize the conformational ensembles and underlying intra- and intermolecular dynamics across the oligomerization transition. The observed sensitivity to ionic strength and molecular crowding in the self-association landscape suggests potential regulation of eIF4B nanoscopic and mesoscopic behaviors such as driven by protein modifications, binding partners or changes to the cellular environment.
Spatiotemporal analysis of F-actin polymerization with micropillar arrays reveals synchronization between adhesion sites
Authors: Hollander Sarit , Guo Yuanning , Wolfenson Haguy , Zaritsky Assaf
Published: June 2024

We repurposed micropillar-arrays to quantify spatiotemporal inter-adhesion communication. Following the observation that integrin adhesions formed around pillar tops we relied on the precise repetitive spatial control of the pillars to reliably monitor F-actin dynamics in mouse embryonic fibroblasts as a model for spatiotemporal adhesion-related intracellular signaling. Using correlation-based analyses we revealed localized information-flows propagating between adjacent pillars that were integrated over space and time to synchronize the adhesion dynamics within the entire cell. Probing the mechanical regulation, we discovered that stiffer pillars or partial actomyosin contractility inhibition enhances inter-adhesion F-actin synchronization. Our results suggest that adhesions can communicate and highlight the potential of using micropillar arrays as a tool to measure spatiotemporal intracellular signaling.
Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells
Authors: Oqua Affiong I , Chao Kin , El Eid Liliane , Casteller Lisa , Baxter Billy P , Miguéns-Gómez Alba , Barg Sebastian , Jones Ben , Bernadino de la Serna Jorge , Rouse Sarah L , Tomas Alejandra
Published: June 2024

G protein-coupled receptors (GPCRs) are integral membrane proteins which closely interact with their plasma membrane lipid microenvironment. Cholesterol is a lipid enriched at the plasma membrane with pivotal roles in the control of membrane fluidity and maintenance of membrane microarchitecture, directly impacting on GPCR stability, dynamics and function. Cholesterol extraction from pancreatic beta cells has previously been shown to disrupt the internalisation, clustering and cAMP responses of the glucagon-like peptide-1 receptor (GLP-1R), a class B1 GPCR with key roles in the control of blood glucose levels via the potentiation of insulin secretion in beta cells and weight reduction via the modulation of brain appetite control centres. Here, we unveil the detrimental effect of a high cholesterol diet on GLP-1R-dependent glucoregulationin vivo, and the improvement in GLP-1R function that a reduction in cholesterol synthesis using simvastatin exerts in pancreatic islets. We next identify and map sites of cholesterol high occupancy and residence time on activeversusinactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, followed by a screen of key residues selected from these sites and detailed analyses of the effects of mutating one of these residues, Val229, to alanine on GLP-1R interactions with cholesterol, plasma membrane behaviours, clustering, trafficking and signalling in pancreatic beta cells and primary islets, unveiling an improved insulin secretion profile for the V229A mutant receptor. This study 1) highlights the role of cholesterol in regulating GLP-1R responsesin vivo; 2) provides a detailed map of GLP-1R - cholesterol binding sites in model membranes; 3) validates their functional relevance in beta cells; and 4) highlights their potential as locations for the rational design of novel allosteric modulators with the capacity to fine-tune GLP-1R responses.
Point set registration for combining fluorescence microscopy methods
Authors: Severins Ivo , Joo Chirlmin , van Noort John
Published: June 2024

Implementation of combined microscopy methods provides valuable information across various scientific applications. However, aligning the datasets and finding the correct point correspondence poses a challenge, especially for large, randomly distributed point sets that are subject to positional errors and missing points. Here, we provide a three-step procedure to perform point set registration, which can be applied to datasets with millions of points and stays robust even when only 10% of the points correspond. In the first global step, the scaling and rotation parameters for the imaging systems are determined once on a smaller calibration dataset using a geometric hashing algorithm. When the global transformation is known, full experimental datasets can be registered by performing step two: a course registration using cross-correlation, and step three: a precise registration to fine-tune the transformation. After these three steps, point correspondence is determined by setting a distance threshold based on a statistical model of random point sets that additionally provides the matching error. We have demonstrated its successful implementation in coupling fluorescence and sequencing methodologies. To enable wide application of these point set registration and correspondence algorithms we provide a python library called MatchPoint.
Exosomes Released from Senescent Cells and Circulatory Exosomes Isolated from Human Plasma Reveal Aging-associated Proteomic and Lipid Signatures
Authors: Patel Sandip Kumar , Bons Joanna , Rose Jacob P. , Chappel Jessie R. , Beres Rebecca L. , Watson Mark A. , Webster Corey , Burton Jordan B. , Bruderer Roland , Desprez Pierre-Yves , Reiter Lukas , Campisi Judith , Baker Erin S. , Schilling Birgit
Published: June 2024

Senescence emerged as a significant mechanism of aging and age-related diseases, offering an attractive target for clinical interventions. Senescent cells release a senescence-associated secretory phenotype (SASP), including exosomes that may act as signal transducers between distal tissues, propagating secondary or bystander senescence and signaling throughout the body. However, the composition of exosome SASP remains underexplored, presenting an opportunity for novel unbiased discovery. Here, we present a detailed proteomic and lipidomic analysis of exosome SASP using mass spectrometry from human plasma from young and older individuals and from tissue culture of senescent primary human lung fibroblasts. We identified ~1,300 exosome proteins released by senescent fibroblasts induced by three different senescence inducers causing most exosome proteins to be differentially regulated with senescence. In parallel, a human plasma cohort from young and old individuals revealed over 1,350 exosome proteins and 171 plasma exosome proteins were regulated when comparing old vs young individuals. Of the age-regulated plasma exosome proteins, we observed 52 exosome SASP factors that were also regulated in exosomes from the senescent fibroblasts, including serine protease inhibitors (SERPINs), Prothrombin, Coagulation factor V, Plasminogen, and Reelin. In addition, 247 lipids were identified with high confidence in all exosome samples. Following the senescence inducers, a majority of the identified phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin species increased significantly indicating cellular membrane changes. The most notable categories of significantly changed proteins were related to extracellular matrix remodeling and inflammation, both potentially detrimental pathways that can damage surrounding tissues and even induce secondary or bystander senescence. Our findings reveal mechanistic insights and potential senescence biomarkers, enabling a better approach to surveilling the senescence burden in the aging population and offering promising therapeutic targets for interventions.
Engineered yeast multicellularity via synthetic cell-cell adhesion and direct-contact signalling
Authors: Meng Fankang , Shaw William M. , Kam Yui Kei Keith , Ellis Tom
Published: June 2024

Coordination of behaviour in multicellular systems is one the main ways that nature increases the complexity of biological function in organisms and communities. WhileSaccharomyces cerevisiaeyeast is used extensively in research and biotechnology, it is a unicellular organism capable of only limited multicellular states. Here we expand the possibilities for engineering multicellular behaviours in yeast by developing modular toolkits for two key mechanisms seen in multicellularity, contact-dependent signalling and specific cell-to-cell adhesion. MARS (Mating-peptideAnchoredResponseSystem) is a toolkit based on surface-displayed fungal mating peptides and G protein-coupled receptor (GPCR) signalling which can mimic juxtacrine signalling between yeasts. SATURN (SaccharomycesAdhesionToolkit for multicellUlar patteRNing) surface displays adhesion-proteins pairs on yeasts and facilitates the creation of cell aggregation patterns. Together they can be used to create multicellular logic circuits, equivalent to developmental programs that lead to cell differentiation based on the local population. Using MARS and SATURN, we further developed JUPITER (JUxtacrine sensor forProtein-protein InTERaction), a genetic sensor for assaying protein-protein interactions in culture, demonstrating this as a tool to select for high affinity binders among a population of mutated nanobodies. Collectively, MARS, SATURN, and JUPITER present valuable tools that facilitate the engineering of complex multicellularity with yeast and expand the scope of its biotechnological applications.
The structural, functional, and neurophysiological connectome of mild traumatic brain injury: a DTI, fMRI and MEG multimodal clustering and data fusion study
Authors: Zhang Jing , Solar Kevin , Safar Kristina , Zamyadi Rouzbeh , Vandewouw Marlee M. , Da Costa Leodante , Rhind Shawn G. , Jetly Rakesh , Dunkley Benjamin T.
Published: June 2024

The clinical presentation and neurobiology of mild traumatic brain injury (mTBI) - also referred to as concussion - are complex and multifaceted, and interrelationships between neurobiological measures derived from neuroimaging are poorly understood. This study applied machine learning (ML) to multimodal whole-brain functional connectomes from magnetoencephalography (MEG) and functional magnetic resonance imaging (fMRI), and structural connectomes from diffusion tensor imaging (DTI) in a test of discriminative accuracy in cases of mTBI. Resting state MEG (amplitude envelope correlations), fMRI (BOLD correlations), and DTI (fractional anisotropy, FA; streamline count, SC) connectome data was acquired in 26 controls without mTBI (all male; 27.6 ± 4.7 years) and 24 participants with mTBI (all male; 29.7 ± 6.7 years) in the acute-subacute phase of injury. ML with data fusion was used to optimally identify modalities and brain features for discriminating individuals with mTBI from those without. Univariate group differences were only found for MEG functional connectivity, while no differences were found for fMRI or DTI. Functional connectivity (fMRI and MEG) showed robust unimodal classification accuracy for mTBI, followed by structural connectivity (DTI), where FA showed marginally better classification performance than SC, but SC outperformed FA in data interpretation and fusion. Perfect, unsupervised separation of participants with and without mTBI was achieved through participant fusion maps featuring all three data modalities. Finally, the MEG-only full feature fusion map showed group differences, and this effect was eliminated upon integrating DTI and fMRI datasets. The markers identified here align well with prior multimodal findings in concussion and highlight modality-specific considerations for their use in understanding network abnormalities of mTBI.
NeoGx: Machine-Recommended Rapid Genome Sequencing for Neonates
Authors: Antoniou Austin A. , McGinley Regan , Metzler Marina , Chaudhari Bimal P.
Published: June 2024

Background

Genetic disease is common in the Level IV Neonatal Intensive Care Unit (NICU), but neonatology providers are not always able to identify the need for genetic evaluation. We trained a machine learning (ML) algorithm to predict the need for genetic testing within the first 18 months of life using health record phenotypes.

Methods

For a decade of NICU patients, we extracted Human Phenotype Ontology (HPO) terms from clinical text with Natural Language Processing tools. Considering multiple feature sets, classifier architectures, and hyperparameters, we selected a classifier and made predictions on a validation cohort of 2,241 Level IV NICU admits born 2020-2021.

Results

Our classifier had ROC AUC of 0.87 and PR AUC of 0.73 when making predictions during the first week in the Level IV NICU. We simulated testing policies under which subjects begin testing at the time of first ML prediction, estimating diagnostic odyssey length both with and without the additional benefit of pursuing rGS at this time. Just by using ML to accelerate initial genetic testing (without changing the tests ordered), the median time to first genetic test dropped from 10 days to 1 day, and the number of diagnostic odysseys resolved within 14 days of NICU admission increased by a factor of 1.8. By additionally requiring rGS at the time of positive ML prediction, the number of diagnostic odysseys resolved within 14 days was 3.8 times higher than the baseline.

Conclusions

ML predictions of genetic testing need, together with the application of the right rapid testing modality, can help providers accelerate genetics evaluation and bring about earlier and better outcomes for patients.
hubEnsembles: Ensembling Methods in R
Authors: Shandross Li , Howerton Emily , Contamin Lucie , Hochheiser Harry , Krystalli Anna ,   Consortium of Infectious Disease Modeling Hubs, Reich Nicholas G. , Ray Evan L.
Published: June 2024

Combining predictions from multiple models into an ensemble is a widely used practice across many fields with demonstrated performance benefits. The R packagehubEnsemblesprovides a flexible framework for ensembling various types of predictions, including point estimates and probabilistic predictions. A range of common methods for generating ensembles are supported, including weighted averages, quantile averages, and linear pools. ThehubEnsemblespackage fits within a broader framework of open-source software and data tools called the “hubverse”, which facilitates the development and management of collaborative modelling exercises.
Structural basis for the transport and regulation mechanism of the Multidrug resistance-associated protein 2
Authors: Koide Eriko , Pietz Harlan L. , Beltran Jean , Chen Jue
Published: June 2024

Multidrug resistance-associated protein 2 (MRP2) is an ATP-powered exporter important for maintaining liver homeostasis and a potential contributor to chemotherapeutic resistance. Deficiencies in MRP2 function are associated with Dubin-Johnson Syndrome and increased vulnerability to liver injury from cytotoxic drugs. Using cryogenic electron microscopy (cryo-EM), we determined the structures of human MRP2 in three conformational states: an autoinhibited state, a substrate-bound pre-translocation state, and an ATP-bound post-translocation state. These structures show that MRP2 functions through the classic alternating access model, driven by ATP binding and hydrolysis. Its cytosolic regulatory (R) domain serves as a selectivity gauge, wherein only sufficiently high concentrations of substrates can effectively compete with and disengage the R domain to initiate transport. Comparative structural analyses of MRP2 in complex with different substrates reveal how the transporter recognizes a diverse array of compounds, highlighting the transporter’s role in multidrug resistance.
Independent origins of spicules reconcile the evolutionary history of sponges
Authors: Rossi Maria Eleonora , Kenny Nathan James , Giacomelli Mattia , Keating Joseph N. , Alvarez-Carretero Sandra , Schuster Astrid , Cardenas Paco , Taboada Sergio , Koutsouveli Vasiliki , Donoghue Philip , Riesgo Ana , Pisani Davide
Published: June 2024

Sponges (Porifera) are highly effective ecosystem engineers, playing a critical role in global biogeochemical processes, including the nitrogen, carbon, and silica cycles. Because of that, they have been closely linked to the evolution of Earth's environments. However, determining the evolutionary history of sponges has posed challenges. Molecular divergence times have pointed to a Tonian or Cryogenian origin for sponges. This contrasts with the oldest unequivocal fossil evidence for sponges, consisting of isolated siliceous spicules from the latest Ediacaran, resulting in a 150-million-mismatch with molecular clock estimates. This mismatch may arise because molecular clock estimates are inaccurate, the fossil record is incomplete, and/or the fossil record has been misinterpreted in the calibration of molecular clock analyses or some combination of the above. To resolve the deep evolutionary history of sponges we compiled an extensive phylogenomic dataset of sponges, revised fossil calibration evidence to derive a new phylogeny and timescale for sponge evolution, and investigated the evolutionary origin of the sponge spicules. Our results confirm that sponges include two main lineages: Silicea (Hexactinellida plus Demospongiae) and Calcarea plus Homoscleromorpha. Our molecular clock analyses identify an early Ediacaran estimate for the crown ancestor of sponges. Our study of spicule evolution shows that neither the last common sponge ancestor, nor the last common ancestors of Silicea and (Calcarea plus Homoscleromorpha) possessed siliceous spicules, which independently evolved four times in sponges: once in Hexactinellida, twice in Demospongiae, and once in Homoscleromorpha. Furthermore, diversification rate analyses demonstrate that the origin of spicules did not align with species radiations within the phylum. Our dating analyses substantially reduce the mismatch between molecular clock estimates for the antiquity of sponges and their fossil record. Our study of the evolutionary history of siliceous spicules reconciles the remaining difference as a consequence of crown-sponges primitively lacking siliceous spicules. As such we demonstrate the power of combining molecular and palaeontological approaches to establishing evolutionary timescales which together allow us to calibrate genome evolution to geological time and read evolutionary history within the gaps in the fossil record.
Comparative Approaches for Quantification of Product Yield in a Model Recombinant Green Fluorescent Protein Expressed inE. coli
Authors: Rusch Gabrielle , Wang Junhyeong , Breau Keith , Kilgour Katie , Gilleskie Gary , Keele Jeff , Selle Kurt , Magness Scott T. , Menegatti Stefano , Daniele Michael
Published: June 2024

Process Analytical Technologies (PAT) used to monitor and control manufacturing processes are crucial for efficient and automated bioprocessing, which is in congruence with lights-off-manufacturing and Industry 4.0 initiatives. As biomanufacturing seeks to realize more high-throughput and automated operation, an increasing need for multimodal analysis of process metrics becomes essential. Herein, we detail a series of methods for analyzing product yield from a bioreactor and how to conduct cross-method comparisons. We employ a model system ofEscherichia coli(E. coli) expression of green fluorescent protein (GFP), which is a simple, cost effective model for students and educators to replicate at different scales. GFP is an ideal analytical marker as it is easy to visualize due to its fluorescence which indicates cellular protein expression, cell localization and physiological changes of the cell population. In this study, samples from a 300 L bioreactor with GFP-expressingE. coliare analyzed to improve product yield and bioprocessing efficiency. Utilizing a fed-batch process for enhanced cell density and product titer, this bioreactor runs on a 24-hour schedule from inoculation to GFP induction and final harvest. To reliably quantify relative GFP expression andE. coliproliferation, we provide simple protocols and example results for comparing three different analytical methods: (1) in-line bioreactor measurements, (2) plate reader assays, and (3) microscopy. The GFP and cell density results follow similar trends based on the various inline and offline analytical methods and show a peak of GFP expression and cell density between 12.5 and 18 hours post inoculation.
Role of the antiparallel double-stranded filament form of FtsA in activating theEscherichia colidivisome
Authors: Perkins Abbigale , Mounange-Badimi Mwidy Sava , Margolin William
Published: June 2024

The actin-like FtsA protein is essential for function of the cell division machinery, or divisome, in many bacteria includingEscherichia coli. Previousin vitrostudies demonstrated that purified wild-type FtsA assembles into closed mini-rings on lipid membranes, but oligomeric variants of FtsA such as FtsAR286Wand FtsAG50Ecan bypass certain divisome defects and form arc and double-stranded (DS) oligomeric states, respectively, which may reflect conversion of an inactive to an active form of FtsA. Yet, it remains unproven which oligomeric forms of FtsA are responsible for assembling and activating the divisome. Here we used an in vivo crosslinking assay for FtsA DS filaments to show that they largely depend on proper divisome assembly and are prevalent at later stages of cell division. We also used a previously reported variant that fails to assemble DS filaments, FtsAM96E R153D, to investigate the roles of FtsA oligomeric states in divisome assembly and activation. We show that FtsAM96E R153Dcannot form DS filamentsin vivo, fails to replace native FtsA, and confers a dominant negative phenotype, underscoring the importance of the DS filament stage for FtsA function. Surprisingly, however, activation of the divisome through theftsL* orftsW* superfission alleles suppressed the dominant negative phenotype and rescued the functionallity of FtsAM96E R153D. Our results suggest that FtsA DS filaments are needed for divisome activation once it is assembled, but they are not essential for divisome assembly or guiding septum synthesis.

IMPORTANCE

Cell division is fundamental for cellular duplication. In simple cells likeEscherichia colibacteria, the actin homolog FtsA is essential for cell division and assembles into a variety of protein filaments at the cytoplasmic membrane. These filaments help to tether polymers of the tubulin-like FtsZ to the membrane at early stages of cell division, but also play crucial roles in recruiting other cell division proteins to a complex called the divisome. Once assembled, theE. colidivisome subsequently activates synthesis of the division septum that splits the cell in two. One recently discovered oligomeric conformation of FtsA is an antiparallel double stranded filament. Using a combination of in vivo crosslinking and genetics, we provide evidence suggesting that these FtsA double filaments have a crucial role in activating the septum synthesis enzymes.
The evolutionarily conserved PhLP3 is essential for sperm development inDrosophila melanogaster
Authors: Petit Christopher , Kojak Elizabeth , Webster Samantha , Marra Michela , Sweeney Brendan , Chaikin Claire , Jemc Jennifer C. , Kanzok Stefan M.
Published: June 2024

Phosducin-like proteins (PhLP) are thioredoxin domain-containing proteins that are highly-conserved across unicellular and multicellular organisms. PhLP family proteins are hypothesized to function as co-chaperones in the folding of cytoskeletal proteins. Here, we present the initial molecular, biochemical, and functional characterization ofCG4511asDrosophila melanogaster PhLP3. We cloned the gene into a bacterial expression vector and produced enzymatically active recombinant PhLP3, which showed similar kinetics to previously characterized orthologues. A fly strain homozygous for a P-element insertion in the 5’ UTR of thePhLP3gene exhibited significant downregulation ofPhLP3expression. We found these male flies to be sterile. Microscopic analysis revealed altered testes morphology and impairment of spermiogenesis, leading to a lack of mature sperm. Among the most significant observations was the lack of actin cones during sperm maturation. Excision of the P-element insertion inPhLP3restored male fertility, spermiogenesis, and seminal vesicle size. Given the high level of conservation of PhLP3, our data suggests PhLP3 may be an important regulator of sperm development across species.
Evaluation of suitable reference genes for qPCR normalisation of gene expression in a Achilles tendon injury model
Authors: Marr Neil , Meeson Richard , Piercy Richard J. , Hildyard John C. W. , Thorpe Chavaunne T.
Published: June 2024

Tendons are one of the major load-bearing tissues in the body; subjected to enormous peak stresses, and thus vulnerable to injury. Cellular responses to tendon injury are complex, involving inflammatory and repair components, with the latter employing both resident and recruited exogenous cell populations. Gene expression analyses are valuable tools for investigating tendon injury, allowing assessment of repair processes and pathological responses such as fibrosis, and permitting evaluation of therapeutic pharmacological interventions. Quantitative polymerase chain reaction (qPCR) is a commonly used approach for such studies, but data obtained by this method must be normalised to reference genes: genes known to be stably expressed between the experimental conditions investigated. Establishing suitable tendon injury reference genes is thus essential. Accordingly we investigated mRNA expression stability in a rat model of tendon injury, comparing both injured and uninjured tendons, and the effects of rapamycin treatment, at 1 and 3 weeks post injury. We used 11 candidate genes (18S, ACTB, AP3D1, B2M, CSNK2A2, GAPDH, HPRT1, PAK1IP1, RPL13a, SDHA, UBC) and assessed stability via four complementary algorithms (Bestkeeper, deltaCt, geNorm, Normfinder). Our results suggests thatACTB,CSNK2A2,HPRT1andPAK1IP1are all stably expressed in tendon, regardless of injury or drug treatment: any three of these would serve as universally suitable reference gene panel for normalizing qPCR expression data in the rat tendon injury model. We also reveal18S,UBC,GAPDH, andSDHAas consistently poor scoring candidates (with the latter two exhibiting rapamycin- and injury-associated changes, respectively): these genes should be avoided.
Fermentation and its effect on the physicochemical and sensory attributes of cocoa beans in the Colombian Amazon
Authors: Ramírez González Andrés Felipe , Gutiérrez García Gustavo Adolfo , Polanía-Hincapié Paola Andrea , López Luis Javier , Suárez Juan Carlos
Published: June 2024

Cocoa (Theobroma cacaoL.) is the basic raw material to produce chocolate and other derivatives such as cocoa butter, cocoa powder and cocoa liquor (cocoa paste), which requires a fermentation process that affects its chemical composition and sensory profile. The objective of this study was to monitor the biochemical, physical and sensory changes during fermentation of cocoa beans in cocoa bean processing plants in the department of Caquetá, Colombia. During fermentation, the temperature of the mass and the pH of the pulp and beans were monitored at the different cocoa processing plants (sites ASOACASAN ASA, COMICACAO CMI, COMCAP COC). Also, at two points during fermentation (days 4 and 7), physical properties of the bean were determined, such as variables related to bromatological composition, polyphenolic compounds and antioxidant activity as sensory attributes at the different sites. An increase in dough temperature was found, however the pH of the cotyledon decreased during the fermentation process and the fat and moisture content varied with fermentation time. At the site level, total polyphenol content (TPC), total flavonoids (TF), DPPH and FRAP contents were statistically different, with COC being different from the other sites. The TPC was higher at the COC site (507 mg GAE/g Cocoa) with respect to the other sites (< 360 mg GAE/g Cocoa). The TF content followed a similar behavior to TPC, with significant differences between sites and differences between fermentation times for ASA. The TF was higher in COC (309.1 mg Catechin/g cocoa) with respect to CMI (215.6 mg Catechin/g cocoa) and ASA (185.7 mg Catechin/g cocoa). Values in DPPH ranged from 5869.3 to 7781.8 𝜇mol Trolox/g cocoa and for the FRAP assay ranged from 369.8 to 606.7 mg AA/g cocoa among the sites.
Linalool’s Multifaceted Antimicrobial Potential: Unveiling its Antimicrobial Efficacy and Immunomodulatory Role AgainstSaprolegnia parasitica
Authors: Tang Tao , Zhong Weiming , Tang Puyu , Dai Rongsi , Guo Jiajing , Gao Zhipeng
Published: June 2024

Saprolegnia parasitica(S.parasitica) is one of the most virulent oomycete species in freshwater aquatic environments, causing severe saprolegniasis and leading to significant economic losses in the aquaculture industry. Thus far, the prevention and control of saprolegniasis face a shortage of medications. Linalool, a natural antibiotic alternative found in various essential oils, exhibits promising antimicrobial activity against a wide range of pathogens. In this study, the specific role of linalool in protectingS.parasiticainfection at bothin vitroandin vivolevels was investigated. Linalool showed multifaceted anti-oomycetes potential by both of antimicrobial efficacy and immunomodulatory efficacy. Forin vitrotest, linalool exhibited strong anti-oomycetes activity and mode of action included: (1) Linalool disrupted the cell membrane of the mycelium, causing the intracellular components leak out; (2) Linalool prohibited ribosome function, thereby inhibiting protein synthesis and ultimately affecting mycelium growth. Surprisingly, meanwhile we found the potential immune protective mechanism of linalool in thein vivotest: (1) Linalool enhanced the complement and coagulation system which in turn activated host immune defense and lysateS.parasiticacells; (2) Linalool promoted wound healing, tissue repair, and phagocytosis to cope withS.parasiticainfection; (3) Linalool positively modulated the immune response by increasing the abundance of beneficial Actinobacteriota; (4) Linalool stimulated the production of inflammatory cytokines and chemokines to lyseS.parasiticacells. In all, our findings showed that linalool possessed multifaceted anti-oomycetes potential which would be a promising natural antibiotic alternative to cope withS.parasiticainfection in the aquaculture industry.
Structure of the E3 ligase CRL2-ZYG11B with substrates reveals the molecular basis for N-degron recognition and ubiquitination
Authors: Liu Xi , Li Yang , Castro Lennice , Yu Zanlin , Cheng Yifan , Daugherty Matthew D. , Gross John D.
Published: June 2024

ZYG11B is a substrate specificity factor for Cullin-RING ubiquitin ligase (CRL2) involved in many biological processes, including Gly/N-degron pathways. Yet how the binding of ZYG11B with CRL2 is coupled to substrate recognition and ubiquitination is unknown. We present the Cryo-EM structures of the CRL2-ZYG11B holoenzyme alone and in complex with a Gly/N-peptide from the inflammasome-forming pathogen sensor NLRP1. The structures indicate ZYG11B folds into a Leucine-Rich Repeat followed by two armadillo repeat domains that promote assembly with CRL2 and recognition of NLRP1 Gly/N-degron. ZYG11B promotes activation of the NLRP1 inflammasome through recognition and subsequent ubiquitination of the NLRP1 Gly/N-degron revealed by viral protease cleavage. Our structural and functional data indicate that blocking ZYG11B recognition of the NLRP1 Gly/N-degron inhibits NLRP1 inflammasome activation by a viral protease. Overall, we show how the CRL2-ZYG11B E3 ligase complex recognizes Gly/N-degron substrates, including those that are involved in viral protease-mediated activation of the NLRP1 inflammasome.
Active fluctuations of axoneme oscillations scale with number of dynein motors
Authors: Sharma Abhimanyu , Friedrich Benjamin M. , Geyer Veikko F.
Published: June 2024

Fluxes of energy generate active forces in living matter, yet also active fluctuations. As canonical example, collections of molecular motors exhibit spontaneous oscillations with frequency jitter caused by non-equilibrium phase fluctuations. We investigate phase fluctuations in reactivatedChlamydomonasaxonemes, which are accessible to direct manipulation. We quantify the precision of axonemal oscillations after controlled chemical removal of dynein motors, providing an experimental test for the theory prediction that the quality factor of motor oscillations should increase with motor number. Our quantification reveals specialized roles of inner and outer arm dynein motors. This supports a model in which inner dyneins serve as master pace-makers, to which outer arm dyneins become entrained, consistent with recent insight provided by structural biology.
The Influence of Ionic Environment on Nucleosome-Mica Interactions Revealed via Molecular Dynamics Simulations
Authors: Kariyawasam Nilusha L. , Wereszczynski Jeff
Published: June 2024

Mica serves as a crucial substrate in Atomic Force Microscopy (AFM) studies for visualizing and characterizing nucleosomes. Nucleosomes interact with the negatively charged mica surface via adsorbed cations. However, the specific influences of monovalent and divalent cations on nucleosome adsorption to the mica surface remain unclear. In this study, we investigated the binding of nucleosomes to the mica surface in the presence of monovalent potassium ions and divalent magnesium ions using molecular dynamics simulations. We also explored the impact of pre-treated mica surfaces on nucleosome binding and structure. Our findings reveal that nucleosome-mica interactions vary depending on the cations present, resulting in distinct effects on nucleosome structure. Notably, nucleosomes bind effectively to a mica surface in the presence of potassium ions with minimal structural perturbations.